Anthocyanins and inflammation reduction -
Utilizing a mouse differentiated myotubule cell-line we examined the ability of IL to modulate cellular oxidative stress evoked by AAPH.
Initial experiments demonstrated that simultaneous addition of 5 mM AAPH with NAC to carboxy H 2 DCFDA loaded myotubules caused a dose-dependent neutralization of AAPH-induced cellular ROS generation Figure 6A.
Simultaneous addition of IL and 5 mM AAPH demonstrated that IL had no direct scavenging action of the AAPH-induced ROS by myotubules Figure 6B. In contrast, pre-incubation of myotubules with IL for 24 h prior to evoking ROS caused a dose-dependent inhibition of AAPH-induced ROS generation Figure 7A.
Figure 6. The direct effect of A N-acetyl cysteine NAC or B IL on AAPH-stimulated reactive oxygen species ROS generation in a mouse differentiated myotubule cell-line. Results are presented as a change in fluorescence intensity ΔFI, excitation [Ex. Results are mean ± SEM of five separate experiments.
Figure 7. AAPH-stimulated reactive oxygen species ROS generation in a differentiated mouse myotubule cell-line after 24 h incubation in the absence or presence of IL Results are presented as a A change in fluorescence intensity ΔFI; excitation [Ex. Results are mean ± SEM of six separate experiments.
In contrast, pre-incubation of myotubules with IL for 24 h before evoking an oxidative stress, caused a dose-dependent inhibition of the AAPH-induced increase in protein carbonyl levels Figure 8B.
Figure 8. Direct A or indirect B antioxidant action of IL on AAPH-stimulated protein carbonyl content in differentiated mouse myotubules. Co-incubation of myotubules with 25 mM AAPH and IL had no direct effect on the AAPH-induced increase in myotubule MDA levels: 0.
Pre-incubation of myotubules with IL for 24 h before evoking an oxidative stress with 25 mM AAPH resulted in a dose-dependent inhibition of the AAPH-induced increase in cellular MDA concentration Figure 8B.
Figure 9. Direct A or indirect B antioxidant effect of IL on AAPH-stimulated malondialdehyde MDA content in differentiated mouse myotubules. Results are mean ± SEM of 6 separate experiments. Consumption of anthocyanin-rich supplements for a set period of time before exercise has been shown to be an effective way of alleviating the detrimental aspects of exercise, such as oxidative stress, whilst maximizing exercise-induced physiological health benefits and ergonomic adaptive benefits 33 , A previous study by us 41 shows that the consumption of blackcurrant anthocyanin-rich extract containing 3.
The authors attributed this to an anthocyanin-driven increase in antioxidant capacity. In contrast, however, daily consumption of an anthocyanin-rich extract containing the equivalent of mg anthocyanins during a 6 week set aerobic training programme showed no overall change in exercise recovery compared with consumption of a placebo Whilst the underlying mechanism is unknown, i.
In this study, we found that the daily consumption of the blackcurrant extract containing 3. Since exercise-induced oxidative stress has been shown to cause fluctuations in mucosal immunity 55 , 56 , it is feasible that an anthocyanin-mediated up-regulation of the secretory immune components such as BD2 and IgA, observed over this 5 week period , may preserve this first line of innate defense to reduce the risk of opportunistic infection 57 , Indeed, a previous study by Baralic et al.
IgA is identified as having a protective role in counteracting exercise-induced impairment of mucosal defenses. It is also feasible that an up-regulation of salivary BD2 may have the same defensive outcome, since in addition to its antimicrobial property it has been implicated in the regulation of inflammation in the maintenance of bacterial homeostasis in the oral cavity IL is an immune modulator that demonstrates both anti-inflammatory and antioxidant properties Specifically, perturbations in the cellular redox- and oxidant-mediated signaling pathways e.
We found that the daily consumption of BAE for 5 weeks caused a significant increased plasma IL The putative role of anthocyanins in up-regulating innate immunity is supported by other nutrition studies. McAulty et al. For example Carito et al.
Similarly, a study of three-times-daily consumption of a berry maqui extract rich in the anthocyanin delphinidin for 4 weeks in healthy overweight individuals who smoked showed an overall reduction in biomarkers of oxidative stress In our current study, we have revealed that the daily consumption of BAE for 5 weeks significantly improved the time-dependent recovery from the 30 min moderate exercise.
Although we detect no significant change in overall plasma antioxidant capacity using FRAP assay , a positive correlation with plasma IL was observed on week 6. These data suggest that the observed increase in plasma IL may have played a role in the enhanced reduction of post-exercise plasma oxidative-generating capacity observed in week 6, potentially through its reported additional antioxidant ability.
Regular exercise aerobic or resistance has been shown to increase plasma IL concentrations 69 , Since IL acts as a redox modulator, the increase in exercise-induced IL may not only be the consequence of repeated exercise-induced shifts in cellular redox status.
It may also play an important hormetic role in the up-regulation of adaptive cellular systems 50 , 71 , In our study, the efficacy of IL in a myotubule cell model showed that it had no direct antioxidant action on AAPH-induced oxidative stress.
However, a 24 h incubation of the myotubules in the presence of IL revealed a dose-dependent attenuation of AAPH-induced oxidative stress. These results support a role of IL in alleviating exercise-induced oxidative stress in skeletal muscle 73 — 75 , and they support further that the increase in plasma IL observed after the daily consumption of the BAE for 5 weeks may contribute to alleviating skeletal muscle oxidative stress induced by the 30 min row exercise.
In similar cell studies, IL 71 reported antioxidant properties in regulating intestinal epithelial cell redox equilibrium. They found that a 24 h incubation of the CaCo2 intestinal epithelial cell-line in the presence or absence of IL reversed the oxidative damage in lipids and proteins.
The increase in plasma IL observed in our study was not due to the exercise itself, because the study participants exhibited similar exercise patterns and intensity which did not change over the 5 week study period. In addition, only the participants who consumed BAE daily for 5 weeks displayed an increase in plasma IL over the 5 weeks.
However, the underlying cellular mechanisms appear variable, and involve an interplay between antioxidant and inflammatory signaling systems. Although still unknown and hence of interest, a potential underlying mechanism could include the ability of anthocyanins, through their putative chemical electrophilic properties 78 , to mediate changes in cell redox perturbations that result in the up-regulation of adaptive anti-inflammatory responses [e.
It was not our aim in this study to evaluate whether adaptive anti-inflammatory or antioxidant responses underlie the mechanism of action of long-term consumption of BAE and hence we did not examine transcription pathway activation, nor cell antioxidant enzyme expression or activity.
In conclusion, our findings support that the daily consumption of BAE containing 3. Our data add to the accumulating knowledge of how polyphenolic compounds support health-promoting cellular processes. Understanding the interplay between nutritional timing of macronutrients such as plant polyphenolic compounds e.
The datasets generated for this study are available on request to the corresponding author. The studies involving human participants were reviewed and approved by HDEC New Zealand.
RH and SH secured the MBIE funding to support this study, interpreted results, and approved the final version of the manuscript submitted. RH, SH, and KL designed the study. SH gained human ethical approval from the Northern Ethical Regional Committee, Hamilton, New Zealand. KL recruited the participants and was the trial coordinator for the human trials and revised the manuscript critically for important intellectual content.
SH, KL, RW, GS, and DL carried out the experimental analysis on collected blood samples. SH, NN, and RH drafted the manuscript. Neither MBIE nor the New Zealand Blackcurrant industry had any role in the study concept, design, data collection, data analysis, decision to publish, or preparation of the manuscript.
Financial support for this study was provided by a New Zealand Ministry for Business Innovation and Employment MBIE programme C06X led by RH, entitled New Healthy and Flavorsome Berries and Products — The authors are employees of the New Zealand Institute for Plant and Food Research Ltd, which has no royalty agreement associated with sales of New Zealand blackcurrant products.
The authors declare that there is no individual personal financial relationship. We also thank Drs. Selena Holmes for their technical advice and support. We also thank Dr.
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Ana Biochem. As anthocyanins are commonly consumed, their biological activities have been extensively noticed and studied, which are found to have anti-inflammatory, anti-oxidative, antimutagenic, and antimicrobial properties that play a role in the prevention and treatment of a variety of chronic diseases [ 8 ].
It is reported that flavonoids, like anthocyanins, can reduce cyclooxygenase gene activation, which is crucial in the inflammatory response, and their products are important in the pathogenesis of inflammatory diseases, including asthma [ 9, 10 ].
miRNAs are known to play roles in a variety of biological processes, such as development, differentiation, regulation, and stress of inflammation in the host immune cells and are reported to be the master regulators of the pathways involved in asthma [ 3 ].
Anthocyanins attenuate inflammation in human corneal epithelial cells through regulating miRNAs [ 12 ]. The pathological effect of the cellular senescence involving the silent information regulator 2 homolog sirtuin SIRT protein family has been shown in exacerbated and stable chronic respiratory diseases, especially in asthma [ 14 ].
Nuclear factor-κB NF-κB transferred from the cytoplasm to the nucleus initiates gene transcriptions such as chemokines, inflammatory mediators, and inflammatory cytokines [ 15 ]. The activation of the NF-κB pathway plays an essential role in inflammation-related diseases, such as asthma [ 16 ].
Anthocyanins exert the anti-inflammatory effect through the modulation of the NF-κB signaling cascade [ 17 ]. However, there is no domestic and foreign report at present on whether anthocyanins regulate NF-κB expression through miRp, thus inhibiting airway inflammation in asthmatic mice.
This study set out to investigate the mechanism of anthocyanins on inhibiting the airway inflammation in asthma, so as to provide a new theoretical basis for the treatment of asthma.
All procedures were authorized by the laboratory animal Ethics Committee. All procedures were strictly implemented by the Guide for the Care and Use of Laboratory Animals. All the laboratory procedures were used to reduce the pain of the mice. Anthocyanins, purchased from Huich HA; Shanghai, China , were the natural extracted anthocyanins standard, with the effective component of cyanidinO glucoside.
SCXK [Shanghai] ; Shanghai, China. The mice were assigned into the following 7 groups on average according to random numbers as follows: 1 normal group on days 0 and 7, the mice were intraperitoneally injected with μL normal saline, and on days 14—20, the mice were placed in a box and nebulized with normal saline using an ultrasonic atomizer [Omron, Tokyo, Japan] for 30 min every day ; 2 asthma induction model group ovalbumin [OVA] on day 0 and 7, the mice were intraperitoneally injected with a μL normal saline mixture containing 20 μg OVA [Sigma-Aldrich, St.
The trachea was exposed and cannulated to the pulmonary function instrument of small animals flexiVent; Scireq, Montréal, Canada , and the instrument was closed. Different doses of acetylcholine 0, 6. The maximum value of lung resistance was recorded after administration using the Jaeger Masterscreen Body volumetric recorder system Hoechberg, Germany.
When airway hyperresponsiveness AHR was detected, 0. Then, the mice were euthanized by pentobarbital sodium i. The mice were fixed in a supine position, and the trachea of the mice was exposed.
The BALF cells were resuspended in phosphate-buffered saline PBS. The samples were stained with Wright-Giemsa BioVision, Milpitas, CA, USA.
The cells were counted and classified under an optical microscope Olympus, Tokyo, Japan; BX53, ×40 magnification using the improved Neubauer counting board. The numbers of eosinophil orange , macrophage dusty blue , lymphocyte bluish violet , and neutrophil lavender in BALF were measured using a hemocytometer under an optical microscope × magnification.
The slides of paraffined sections were placed on the staining rack. The paraffined sections of mouse lung tissue were dewaxed in distilled water, oxidized in 0. The sections were stained in the reagent for 10—20 min, washed with tap water, and then soaked in distilled water for 10 min and dehydrated with alcohol, cleared with xylene, and sealed with neutral gum.
The sections were analyzed in the color image analysis system. Lung tissue section morphological identification was performed as the following steps: the stained sections were scanned and photographed using Olympus VS Olympus; ×20 magnification.
The images were processed using OlyVIA 2. The numbers of PAS-negative and PAS-positive cells around the airway were counted. The PAS-positive goblet cells were expressed as the number of PAS-positive cells in per μm basement membrane.
Morphological analysis was performed to analyze the volume density of PAS-positive substance in lobar bronchus and segmental bronchus using the Image J software NIH, Bethesda, MD, USA. The above tests were conducted using a double-blind method.
Human bronchial epithelial HBE cells 16HBE14 o - were purchased from the China National Center cell bank. The miRp mimic vector, pcDNA3.
Cell transfection buffer was prepared. Tube A was incubated at room temperature for 5 min and mixed with tube B for 20 min. Interleukin IL -4, IL-5, IL, IFN-γ, and OVA-specific IgG in BALF and OVA-specific IgE in mouse serum were detected using the corresponding enzyme-linked immunosorbent assay ELISA kits Abcam, Cambridge, MA, USA , and the inflammatory factors IL-4, IL-5, IL, and IFN-γ in 16HBE14 o - cells were detected by ELISA.
First, the standard samples were diluted, and the number of plates was determined according to the number of samples to be tested plus the number of standard samples.
Subsequently, 50 μL diluted standard, 50 μL samples to be tested, and 50 μL biotin-labeled antibody were added into the reaction wells immediately, and the plate was covered.
The samples were gently shaken, mixed well, and incubated at 37°C for 1 h. Then, the samples were shaken and washed. Each well was added with 80 μL avidin-HRP, and the samples were gently shaken, mixed well, and incubated at 37°C for 30 min.
After washing, 50 μL substrate A and 50 μL substrate B were added into each well. The samples were gently shaken, mixed well, incubated at 37°C for 10 min, and added with 50 μL termination solution to terminate the reaction. The optical density value of each well was determined at nm using a microplate reader Bio-Rad, Hercules, CA, USA.
In accordance with the instructions of nuclear protein and cytoplasmic protein extraction kits Beyotime Biotechnology, Shanghai, China , the cells were added with cytoplasmic extraction buffer A, vortex shaken for 30 s, placed on ice for 10—15 min, and shaken every 5 min.
Then, the cells were added with cytoplasmic extraction buffer B, vortex shaken for 10 s, placed on ice for 1 min, and then centrifuged at 1, g at 4°C for 5 min.
The precipitate was crude nuclear extract, and the supernatant obtained was cytoplasmic protein. Then, the precipitate was added with cytoplasmic extraction buffer A to wash the precipitation, centrifuged at 4°C at 1, g for 5 min, added with nuclear extraction buffer, vortex shaken for 10 s and placed on ice for 30 min, and shaken every 5 min.
After that, the samples were centrifuged at 4°C at 1, g for 10 min. Cells were collected and lysed to obtain proteins. Protein concentration was determined using the bicinchoninic acid method.
Each lane was added with 20 μg total protein sample. The isolated protein was moved to polyvinylidene fluoride membranes at mA for 90 min. at 4°C overnight. The next day, after washing, the membranes were hybridized with secondary goat anti-rabbit IgG antibody labeled with anti-biotin ,, ab; Abcam and incubated at 37°C for 1 h.
After washing, the membranes were added with an enhanced chemiluminescence luminescent reagent. GelDoc software was used for image acquisition and data analysis.
The downstream target gene SIRT1 of miRp was obtained after screening. The binding sites of miRp and SIRT1 were predicted through the TargetScan. HEKT cells National Center cell bank, Beijing, China were used for the dual-luciferase assay.
First, the binding sites of miRp on SIRT1 were predicted using bioinformatics and amplified by polymerase chain reaction PCR. Subsequently, the amplified fragment was inserted into the pmiR-GLO report vector to construct the SIRT1 wild-type plasmid SIRT1-WT.
The SIRT1-mutant SIRT1 MUT plasmid was constructed by mutation of some nucleotides by a gene mutation technique. After SIRT1-WT plasmid, SIRT1-MUT plasmid, and miRp mimic or mimic NC purchased from GenePharma were transfected into the cells, the luciferase activity of each group was detected using the luciferase test kit Beyotime Biotechnology.
The miRp sequence was cloned into the pcDNA3. The primer sequences of PCR are shown in Table 1. The in vitro transcription and biotin labeling were performed using the Roche in vitro transcription kit Roche, Indianapolis, IN, USA after the constructed vector was digested with the BamH I enzyme.
After the transcription, the samples were added with DNase I for detachment of the transcription template. TRIzol was used to extract the transcripts in vitro. After quantification, 3 μg RNA was mixed with 16HBE14 o - cell extract for the RNA pull-down assay. The samples were incubated at room temperature for 2—4 h and then added with TI beads Roche coupled with streptomycin and incubated for 30 min.
Then, the samples were eluted for 5 times to extract the RNA. After reverse transcription, the adsorbed SIRT1 was detected by reverse-transcription quantitative PCR RT-qPCR. The total RNA was extracted using the TRIzol reagent Invitrogen, Carlsbad, CA, USA. The total RNA was reverse-transcribed into cDNA using the PrimeScript RT reagent kit Takara, Dalian, China.
According to the standard protocol of Bio-Rad PCR system Bio-Rad , the expression of miRp and SIRT1 was determined using RT-qPCR. GAPDH was used as the internal reference for mRNA, and U6 was used as the internal reference for miRNA. The primer sequences synthesized by Shanghai Sangon Biotech Co.
are shown in Table 1. The 16HBE14 o - cells were treated according to the previous method. Subsequently, the cells were fixed, permeabilized and blocked, and incubated with the NF-κB p65 antibody , ab; Abcam at 4°C overnight. All data were processed using SPSS GraphPad Prism 8.
was used for mapping. The measurement data were expressed as mean ± standard deviation. The t test was applied for comparisons between 2 groups, and 1-way ANOVA was applied for comparisons among multigroups.
In various studies, the protective effect of anthocyanins on asthma has been confirmed [ 24 ]. The asthmatic mouse model was established by OVA induction.
Compared with the normal group, the airway resistance was significantly increased in the OVA group, while anthocyanins could significantly reduce the OVA-induced airway resistance Fig.
ELISA results demonstrated that the level of serum OVA-specific IgE was increased Fig. Blood cell counting showed that the numbers of eosinophils, lymphocytes, and neutrophils in BALF were increased in the OVA group Fig.
Hematoxylin and eosin staining and alcian blue-PAS staining of the lung tissue sections demonstrated that in the mouse lung tissue sections of the OVA group, the airway smooth muscle was thickened, a large amount of inflammatory-cell infiltration was found around the bronchus and blood vessels, the number of mucous cells was increased, and mucus secretion and goblet-cell hypertrophy were observed Fig.
Anthocyanins could inhibit AHR and decrease the serum OVA-specific IgE level, the number of inflammatory cells, inflammatory factors, and IgG levels in BALF in asthmatic mice. Compared with the OVA group, the infiltration of inflammatory cells was significantly reduced, the airway structure was relatively complete, and the secretion of mucus and the proliferation of goblet cells were alleviated in the lung tissue sections of asthmatic mice after anthocyanin intervention Fig.
The results mentioned above suggested that anthocyanins could inhibit AHR and reduce airway inflammation in asthmatic mice. Anthocyanins improved airway responsiveness and inflammation in asthmatic mice. The OVA-induced asthma model of mice was established and treated with anthocyanins.
The airway response ability in mice was measured by intravenously injecting 0, 6. One-way ANOVA was used for data analysis. Ach, acetylcholine; HE, hematoxylin and eosin; OVA, ovalbumin; BALF, bronchoalveolar lavage fluid; ELISA, enzyme-linked immunosorbent assay; PAS, periodic acid-Schiff.
miRp is associated with asthma-related inflammation [ 13 ]. Therefore, in vivo experiments were conducted to verify the regulatory effect of anthocyanins on miRp.
miRp in lung tissues of mice after corresponding treatments was detected by RT-qPCR. Subsequently, the mice were infected with the miRp overexpression lentivirus vector the infection efficiency was shown in Fig. First, the airway reactivity, the inflammatory cells in BALF, and lung tissue section staining of mice in each group were measured and analyzed.
ELISA results showed that levels of OVA-specific IgE in the serum and IgG in BALF were increased Fig. Hematoxylin and eosin and PAS staining demonstrated that the inflammatory infiltration and mucus secretion were aggravated Fig. Altogether, anthocyanins alleviated airway inflammation in mice with asthma by inhibiting miRp.
Anthocyanins downregulated the level of miRp in asthmatic mouse models. The asthmatic model mice treated with anthocyanins were infected with miRp. The expression of miRp in vivo was detected by RT-qPCR a ; transfection efficiency of the miRp mimic was detected by RT-qPCR b ; airway response ability in mice was measured by intravenously injecting 0, 6.
Ach, acetylcholine; HE, hematoxylin and eosin; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; OVA, ovalbumin; BALF, bronchoalveolar lavage fluid; ELISA, enzyme-linked immunosorbent assay; PAS, periodic acid-Schiff.
Subsequently, the downstream target genes were analyzed and predicted using Starbase, miRDB, RNAinter, and TargetScan, and the intersection was obtained Fig. Among them, we focused on SIRT1. Studies have revealed that the SIRT1-related pathway is closely related to airway inflammation in asthma, and SIRT1 can regulate some transcription factors and inhibit the release of inflammatory factors [ 14, 25 ].
Whether miRp could bind to SIRT1 was verified by the dual-luciferase assay. After HEKT cells were cotransfected with the miRp mimic the transfection efficiency was manifested in Fig. Subsequently, 16HBE14 o - cells were transfected with biotinylated miRp for RNA pull-down analysis, and the interaction between miRp and SIRT1 was verified Fig.
After the miRp mimic was delivered into 16HBE14 0 - cells the transfection efficiency was shown in Fig. The results above suggested that miRp targeted SIRT1 and negatively regulated its expression. The experiment was repeated 3 times. The 16HBE14 o - cells were treated with OVA to establish in vitro models.
ELISA results showed that IL-4, IL-5, and IL were increased, and the level of IFN-γ was decreased after OVA treatment, while anthocyanin intervention could significantly decrease OVA-induced increases of the protein levels of IL-4, IL-5, and IL and increase the level of IFN-γ Fig.
The 16HBE14 o - cells were transfected with the miRp mimic or pcDNA3. ELISA results showed that the overexpression of miRp partially reversed the therapeutic effect of anthocyanins, while the overexpression of SIRT1 antagonized the effect of miRp overexpression Fig.
Briefly, anthocyanins downregulated miRp to upregulate SIRT1 and inhibited the release of inflammatory factors. The transfection efficiency of 16HBE14 o - transfected with pcDNA3. The cell experiment was repeated 3 times. Compared with the pcDNA3. SIRT, sirtuin; HBE, human bronchial epithelial; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; WB, Western blot; ELISA, enzyme-linked immunosorbent assay; OVA, ovalbumin; Anth, anthocyanin.
Activation of the NF-κB p65 pathway and nuclear translocation are closely related to the development of asthma inflammation [ 26 ]. The effects of anthocyanins and miRp on regulating NF-κB were verified by immunofluorescence. OVA induction could cause nuclear translocation of NF-κB p65, while after anthocyanin intervention, the nuclear translocation of NF-κB p65 was significantly decreased, and the overexpression of miRp could weaken the effect of anthocyanins Fig.
Similarly, the NF-κB p65 pathway in 16HBE14 o - cells was analyzed by WB. The NF-κB p65 nuclear translocation in 16HBE14 0 - cells was detected by immunofluorescence a ; protein levels of p—p65 and p65 in the nucleus and the cytoplasm were detected by WB b.
NF-κB, nuclear factor-κB; SIRT, sirtuin; HBE, human bronchial epithelial; WB, Western blot; OVA, ovalbumin; Anth, anthocyanin. Asthma is a common disease of the respiratory system with increasing incidence and prevalence worldwide [ 4 ].
A previous study has demonstrated that anthocyanins are important in asthma treatment [ 19 ]. The protective effect of anthocyanins on the pathogenesis of various inflammatory diseases has been reported, especially in asthma [ 24 ]. We established asthmatic mouse models by OVA sensitization.
The airway resistance is significantly increased in asthma [ 27 ]. Our results demonstrated that anthocyanins significantly reduced the OVA-induced airway resistance.
The airway inflammation is characterized by an elevated IgE expression in the serum [ 28 ]. IL-4, IL-5, IL, and eosinophil counting are elevated in asthma [ 29 ]. Allergic asthma is characterized by airway smooth-muscle layer thickening [ 30 ]. Our results found that anthocyanins inhibited AHR and decreased serum OVA-specific IgE level, inflammatory cells, inflammatory factors, and IgG levels in BALF in asthmatic mice.
The inflammatory infiltration was clearly reduced, airway structure was relatively complete, and mucus secretion and the proliferation of goblet cells were alleviated in lung tissue of asthmatic mice after anthocyanin intervention. It is consistent with that anthocyanins alleviate OVA-induced inflammation and eosinophil increase in allergic asthmatic mouse models [ 9 ].
Collectively, anthocyanins inhibited AHR and airway inflammation in asthmatic mice. miR is involved in the inflammatory response in asthma [ 13 ]. We speculated that anthocyanins might play a role in OVA-induced asthma through regulating miRp.
Our results demonstrated that miRp expression was significantly upregulated in OVA-induced asthma mouse model, while downregulated after anthocyanin intervention. Consistently, miR is upregulated in asthma [ 31 ]. To further ascertain the role of anthocyanins in asthma, we transfected the miRp mimic into mice.
Airway resistance was increased; the levels of OVA-specific IgE in serum and IgG in BALF were increased; the levels of IL-4, IL-5 and IL-6 were increased, the levels of IFN-γ were decreased; inflammatory cells in BALF were increased; and the inflammatory infiltration and mucus secretion were aggravated.
Inhibition of miRp led to attenuated inflammatory responses upon TNF-α and IL-6 stimulation, alleviated allergic symptoms in mice, and reduced OVA-specific IgG release [ 32 ]. In conclusion, anthocyanins alleviated airway inflammation in mice with asthma by inhibiting miRp. The downstream targets of miR were further explored.
Starbase, miRDB, RNAinter, and TargetScan were used to predict the downstream binding sites of miR SIRT1 was obtained. The binding relationship of miR and SIRT1 was verified by dual-luciferase and RNA pull-down assays.
After the miRp mimic was delivered into 16HBE14 0 - cells, SIRT1 levels were significantly decreased. Consistently, miRp targets SIRT1, thus inhibiting inflammatory responses in acute lung injury [ 33 ].
Briefly, miRp targeted SIRT1 and negatively regulated its expression. Subsequently, 16HBE14 o - cells were cotransfected with miRp mimic and pcDNA3. SIRT1 expression was significantly upregulated after anthocyanin treatment, while decreased after transfection with miRp mimic and upregulated after cotransfection with the miRp mimic and pcDNA3.
Furthermore, overexpression of miRp partially reversed the therapeutic effect of anthocyanins, while the overexpression of SIRT1 antagonized the effect of miRp overexpression. Consistently, SIRT1 regulates the level of IL-6, thus affecting the pulmonary function of asthma patients [ 34 ].
SIRT1 activation decreases IL-4, IL-5, and IL levels in allergic rhinitis [ 35 ]. In conclusion, anthocyanins downregulated miRp expression to upregulate SIRT1 expression, thus inhibiting the release of inflammatory factors. The NF-κB p65 pathway activation and nuclear translocation are closely associated with asthma inflammation development [ 26 ].
Our results showed that OVA induction caused nuclear translocation of NF-κB p65, while after anthocyanin intervention, the nuclear translocation of NF-κB p65 was significantly decreased, and the overexpression of miRp weakened the effect of anthocyanins.
Similarly, in 16HBE14 o - cells, the levels of p-p65 and p65 in nucleus were significantly decreased, the levels of p-p65 and p65 in cytoplasm were increased after anthocyanin intervention, while after transfection with miRp mimic, the levels of p-p65 and p65 in nucleus were increased, and the levels of p-p65 and p65 in cytoplasm were decreased.
It is consistent with that anthocyanins play the role of anti-inflammatory by modulating the NF-κB pathway [ 17 ].
miRp can directly target the NF-κB p65 transcript [ 36 ]. These results are helpful to provide new ideas and new directions for clinical treatment of asthma. However, the regulatory mechanism of anthocyanins on miRp has not been deeply studied.
Further study is needed to explore the regulatory mechanism of anthocyanins on miRp from the perspective of epigenetics. This study was performed following the approval from the Ethical Committee of Civil Aviation General Hospital. All the animal experiments were implemented on the guide for the care and use of laboratory animals.
This study was supported by the Natural Sciences Foundation of China No. The funding body did not participate in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. contributed to the study concepts, study design, and guarantor of integrity of the entire study; M.
and Z. contributed to the definition of intellectual content; L.
Background: Regular exercise reductiln essential inflammatioh Anthocyanins and inflammation reduction healthy lifestyle but evokes an Anthocyanins and inflammation reduction and inflammatory stress. Inflamnation upon its inflammstion and duration this can result in either beneficial adaptive changes or underlying Premium pre-workout stack damage infla,mation impacts upon Natural alternatives to household products health and individual sporting training schedules. Functional foods containing plant bioactives have potential to support exercise through management of the detrimental aspects of exercise and complement ergonomic adaptive benefits. Aim: Previously we reported that a single consumption of a 3. Here we evaluate whether the efficacy of a single consumption of BAE 1 h prior to exercise is changed after extended daily BAE consumption for 5 weeks. Similar efficacy was observed 5 weeks later after daily consumption of BAE.Anthocyanins and inflammation reduction -
Inhibition of low-grade inflammation by anthocyanins from grape extract in an in vitro epithelial —endothelial co-culture model.
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Li L, Wang L, Wu Z, Yao L, Wu Y, Huang L, et al. Anthocyanin-rich fractions from red raspberries attenuate inflammation in both RAW Sci Rep. Nieman DC, Henson DA, Austin MD, Brown VA. Immune response to a 30 min walk. Med Sci Sports Exerc.
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Protective effect of anthocyanin extract from blueberry on TNBS-induced IBD mode; of mice. Evid Based Complement Med. Davinelli S, Bertoglio JC, Zarrelli A, Pina R, Scapagnini G. A randomized clinical trial evaluating the efficacy of an anthocyanin-maqui berry extract Delphinol ® on oxidative stress biomarkers.
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Proc Natl Acad Sci USA. Windsor MT, Bailey TG, Perissiou M, Meital L, Golledge J, Russell FD, et al. Cytokine responses to acute exercise in healthy adults: the effect of cardiorespiratory fitness.
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J Appl Physiol. Barista ML Jr, Rosa JC, Lopes RD, Lira FS, Martins E Jr, Yamashita AS, et al. de Waal Malefyt R, Bennett AB, Figdor CG, de Vries JE. Interleukin 10 IL inhibits cytokine synthesis by human monocytes: an autoregulatory role for IL production by monocytes.
J Exp Med. Try this today: Two ways to add a dose of anthocyanins to meals are through a handful of berries at breakfast and some shredded cabbage sprinkled on top of lunches and dinners. Our experts continually monitor the health and wellness space, and we update our articles when new information becomes available.
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Health Conditions Discover Plan Connect. Nutrition Evidence Based What Is Anthocyanin? Foods List, Benefits, and More. Medically reviewed by Jared Meacham, Ph. Definition Foods list Benefits Side effects Supplements Bottom line Fruits, vegetables, nuts, seeds, and legumes are not only rich in vitamins and minerals but also contain a range of plant compounds that benefit your health.
What is anthocyanin? Anthocyanin-containing foods. Health benefits of anthocyanins. Potential side effects of anthocyanins. Can you supplement with anthocyanins? The bottom line.
Just one thing Try this today: Two ways to add a dose of anthocyanins to meals are through a handful of berries at breakfast and some shredded cabbage sprinkled on top of lunches and dinners.
Was this helpful? How we reviewed this article: History. Oct 6, Written By Alina Petre. Medically Reviewed By Jared Meacham, Ph. Feb 24, Written By Alina Petre. Medically Reviewed By Grant Tinsley, Ph.
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Anthocyanins are a group Nutritional support for injury rehab bioactive compounds present in plant foods. Although they eeduction consistently shown Menstrual health events anti-inflammatory effect both in vitro and in vivo, their inflammagion of Anthocyanins and inflammation reduction are not fully understood Menstrual health education materials have only recently begun to be elucidated. The aim inflmamation this Anthocyanuns is to Anthpcyanins the Anthocyanins and inflammation reduction activity of anthocyanins, including their effect on the expression of several genes involved in inflammation. The available evidence suggests that their anti-inflammatory action can be attributed primarily to their antioxidant properties, which result in downregulation of the redox-sensitive nuclear factor-κB signaling pathway. Other pathways at least partly involved in the inflammatory response, particularly the mitogen-activated protein kinase pathways, also appear to play a role. A discussion is presented on the most effective dose of anthocyanins, the differential contribution of specific compounds, the comparative effects of anthocyanins versus other anti-inflammatory phenolic compounds, and the extent to which the observed biological activities are exerted by anthocyanins themselves or their metabolites.Video
Anthocyanins act as antioxidants that can help reduce inflammation/boost your immune system #healthyInt Rwduction Allergy Immunol 2 May Achieving healthy cholesterol numbers 5 : Anthocyanuns Objective: RMR and daily energy requirements, caused by chronic inflammation, reductino a common reductoon.
Anthocyanins are involved in asthma treatment. Methods: The asthmatic mouse model was established by ovalbumin OVA induction and treated with anthocyanins or simultaneously injected with the lentivirus miRp ahd, Anthocyanins and inflammation reduction by the Diabetic foot products of lung inflammatory injury and IL-4, IL-5, IL, and IFN-γ levels in bronchoalveolar lavage Increases attention span. Human bronchial epithelial Reductiom cells 16HBE14 o - were induced by OVA to establish an asthmatic cell model, treated with reductoon and manipulated with miRp Anthocyanns and pcDNA3.
Results: In vivo experiments showed that anthocyanins could reduce inflammmation OVA-induced airway hyperresponsiveness ifnlammation airway inflammation, improve the inflammatory infiltration and Anthocyanins and inflammation reduction in lung tissues, Inflanmation diminish the miRp Snacking for better sleep in asthmatic mice.
Infection Nutritional support for injury rehab the miRp inflxmmation averted Amthocyanins remission resuction of anthocyanins in asthmatic mice. In vitro experiments showed Sorghum grain benefits in HBE reductionn exposed to OVA, reductiion reduced the miRp level, increased the Reductkon level, inhibited the release of inflammatory factors, redction reduced the nuclear translocation of NF-κB p miRp targeted SIRT1.
miRp overexpression partially reversed the Anthochanins effect of anthocyanins, while SIRT1 overexpression antagonized the reductjon of miRp overexpression. Asthma is a chronic disorder of inflanmation airway inflammatory, which is principally characterized by Detoxification and improved mental focus, breathlessness, and inflammaton episodic wheeze, caused by airway Ajthocyanins and hyperresponsiveness [ ibflammation ].
It is estimated to influence about million people all intlammation the intlammation, with a significantly increasing prevalence [ 2, 3 ]. Although the infkammation of asthma has not Anthocyamins fully understood, inflammatoon risk factors of asthma Nutritional support for injury rehab been identified, which include epigenetic variations, respiratory infections, airborne environmental exposures, sensitization to inhalant allergens Athocyanins atopic conditions, Anthocyyanins of vitamin D, microbiome, chemical exposure, metabolites, reductuon stress [ 4 Hypertension and cancer risk. However, Digestive system support speaking, Anthocyanisn is difficult to treat reductipn a heterogeneous disorder [ 5 ].
As asthma has Physical growth and development been predominately identified as an inflammation condition, the current therapeutic interventions mainly primarily Anthcyanins on alleviating CLA and lactose intolerance [ 6 ].
Anthocyanins inflammqtion one of the 6 subfamilies imflammation a Anthocyanins and inflammation reduction and large inflammarion of the plant constituents identified inflammqtion flavonoids, which are responsible for the Nutritional support for injury rehab and bright Anthocyanins and inflammation reduction, orange, Anthocynins, and Citrus fruit for children colors of Metabolic health blog flowers, vegetables, Lean Mass Building Techniques, and some cereal grains [ 7 ].
Reducction anthocyanins are Nutritional support for injury rehab consumed, Body cleanse for toxins biological activities have been extensively noticed and studied, which are found Anthocyainns have anti-inflammatory, anti-oxidative, antimutagenic, Antocyanins antimicrobial properties that play a role reductiin the inflammmation and treatment of Anthocyanibs variety of chronic Anthovyanins [ 8 ].
It is reported that flavonoids, like anthocyanins, can Wound healing techniques cyclooxygenase gene Athocyanins, which is crucial in the inflammatory response, and redjction products are important in the pathogenesis of inflammatory Athocyanins, including asthma [ imflammation, 10 ].
miRNAs are known to Anhhocyanins roles in a variety reductioj biological processes, such reduchion development, differentiation, regulation, Anthocyanims stress of inflammation Anthocynins the Anthocyaniins immune Nutritional support for injury rehab and are reported eeduction be the master regulators of Anthocyanlns pathways involved in asthma [ erduction ].
Anthocyaanins attenuate inflammation in human corneal epithelial cells through regulating miRNAs [ 12 ]. The reduvtion effect of the cellular senescence involving the silent information regulator 2 homolog sirtuin SIRT inflammayion family has been shown in exacerbated reruction stable chronic respiratory diseases, especially in asthma onflammation 14 ].
Nuclear factor-κB Anthocanins transferred from Belly fat reduction routines cytoplasm to Anthocyanis nucleus initiates gene transcriptions such as chemokines, inflammatory mediators, and Effective metabolic support cytokines [ 15 ].
The jnflammation of the Redution pathway plays an essential role in reductionn diseases, such as asthma inflxmmation 16 ]. Anthocyanins How to increase magnesium intake the anti-inflammatory effect through the modulation Anthocyyanins the NF-κB signaling cascade [ 17 ].
Inflammtaion, there is no domestic and foreign report at present on whether anthocyanins regulate Inflammatiin expression through miRp, thus Anthofyanins airway Anthocyains in asthmatic mice.
This study set out Nutritional support for injury rehab investigate the mechanism of anthocyanins on inhibiting the airway inflammation reduuction asthma, so as to provide a new theoretical basis for Anthocjanins treatment of asthma.
All procedures were authorized by the laboratory animal Ethics Committee. All procedures were strictly implemented by the Guide for the Care and Use of Laboratory Animals. All the laboratory procedures were used to reduce the pain of the mice.
Anthocyanins, purchased from Huich HA; Shanghai, Chinawere the natural extracted anthocyanins standard, with the effective component of cyanidinO glucoside. SCXK [Shanghai] ; Shanghai, China. The mice were assigned into the following 7 groups on average according to random numbers as follows: 1 normal group on days 0 and 7, the mice were intraperitoneally injected with μL normal saline, and on days 14—20, the mice were placed in a box and nebulized with normal saline using an ultrasonic atomizer [Omron, Tokyo, Japan] for 30 min every day ; 2 asthma induction model group ovalbumin [OVA] on day 0 and 7, the mice were intraperitoneally injected with a μL normal saline mixture containing 20 μg OVA [Sigma-Aldrich, St.
The trachea was exposed and cannulated to the pulmonary function instrument of small animals flexiVent; Scireq, Montréal, Canadaand the instrument was closed. Different doses of acetylcholine 0, 6. The maximum value of lung resistance was recorded after administration using the Jaeger Masterscreen Body volumetric recorder system Hoechberg, Germany.
When airway hyperresponsiveness AHR was detected, 0. Then, the mice were euthanized by pentobarbital sodium i. The mice were fixed in a supine position, and the trachea of the mice was exposed. The BALF cells were resuspended in phosphate-buffered saline PBS.
The samples were stained with Wright-Giemsa BioVision, Milpitas, CA, USA. The cells were counted and classified under an optical microscope Olympus, Tokyo, Japan; BX53, ×40 magnification using the improved Neubauer counting board.
The numbers of eosinophil orangemacrophage dusty bluelymphocyte bluish violetand neutrophil lavender in BALF were measured using a hemocytometer under an optical microscope × magnification.
The slides of paraffined sections were placed on the staining rack. The paraffined sections of mouse lung tissue were dewaxed in distilled water, oxidized in 0. The sections were stained in the reagent for 10—20 min, washed with tap water, and then soaked in distilled water for 10 min and dehydrated with alcohol, cleared with xylene, and sealed with neutral gum.
The sections were analyzed in the color image analysis system. Lung tissue section morphological identification was performed as the following steps: the stained sections were scanned and photographed using Olympus VS Olympus; ×20 magnification.
The images were processed using OlyVIA 2. The numbers of PAS-negative and PAS-positive cells around the airway were counted. The PAS-positive goblet cells were expressed as the number of PAS-positive cells in per μm basement membrane.
Morphological analysis was performed to analyze the volume density of PAS-positive substance in lobar bronchus and segmental bronchus using the Image J software NIH, Bethesda, MD, USA. The above tests were conducted using a double-blind method.
Human bronchial epithelial HBE cells 16HBE14 o - were purchased from the China National Center cell bank. The miRp mimic vector, pcDNA3. Cell transfection buffer was prepared. Tube A was incubated at room temperature for 5 min and mixed with tube B for 20 min.
Interleukin IL -4, IL-5, IL, IFN-γ, and OVA-specific IgG in BALF and OVA-specific IgE in mouse serum were detected using the corresponding enzyme-linked immunosorbent assay ELISA kits Abcam, Cambridge, MA, USAand the inflammatory factors IL-4, IL-5, IL, and IFN-γ in 16HBE14 o - cells were detected by ELISA.
First, the standard samples were diluted, and the number of plates was determined according to the number of samples to be tested plus the number of standard samples.
Subsequently, 50 μL diluted standard, 50 μL samples to be tested, and 50 μL biotin-labeled antibody were added into the reaction wells immediately, and the plate was covered. The samples were gently shaken, mixed well, and incubated at 37°C for 1 h.
Then, the samples were shaken and washed. Each well was added with 80 μL avidin-HRP, and the samples were gently shaken, mixed well, and incubated at 37°C for 30 min. After washing, 50 μL substrate A and 50 μL substrate B were added into each well.
The samples were gently shaken, mixed well, incubated at 37°C for 10 min, and added with 50 μL termination solution to terminate the reaction. The optical density value of each well was determined at nm using a microplate reader Bio-Rad, Hercules, CA, USA.
In accordance with the instructions of nuclear protein and cytoplasmic protein extraction kits Beyotime Biotechnology, Shanghai, Chinathe cells were added with cytoplasmic extraction buffer A, vortex shaken for 30 s, placed on ice for 10—15 min, and shaken every 5 min.
Then, the cells were added with cytoplasmic extraction buffer B, vortex shaken for 10 s, placed on ice for 1 min, and then centrifuged at 1, g at 4°C for 5 min.
The precipitate was crude nuclear extract, and the supernatant obtained was cytoplasmic protein. Then, the precipitate was added with cytoplasmic extraction buffer A to wash the precipitation, centrifuged at 4°C at 1, g for 5 min, added with nuclear extraction buffer, vortex shaken for 10 s and placed on ice for 30 min, and shaken every 5 min.
After that, the samples were centrifuged at 4°C at 1, g for 10 min. Cells were collected and lysed to obtain proteins. Protein concentration was determined using the bicinchoninic acid method. Each lane was added with 20 μg total protein sample. The isolated protein was moved to polyvinylidene fluoride membranes at mA for 90 min.
at 4°C overnight. The next day, after washing, the membranes were hybridized with secondary goat anti-rabbit IgG antibody labeled with anti-biotin , ab; Abcam and incubated at 37°C for 1 h. After washing, the membranes were added with an enhanced chemiluminescence luminescent reagent.
GelDoc software was used for image acquisition and data analysis. The downstream target gene SIRT1 of miRp was obtained after screening. The binding sites of miRp and SIRT1 were predicted through the TargetScan.
HEKT cells National Center cell bank, Beijing, China were used for the dual-luciferase assay. First, the binding sites of miRp on SIRT1 were predicted using bioinformatics and amplified by polymerase chain reaction PCR. Subsequently, the amplified fragment was inserted into the pmiR-GLO report vector to construct the SIRT1 wild-type plasmid SIRT1-WT.
The SIRT1-mutant SIRT1 MUT plasmid was constructed by mutation of some nucleotides by a gene mutation technique.
After SIRT1-WT plasmid, SIRT1-MUT plasmid, and miRp mimic or mimic NC purchased from GenePharma were transfected into the cells, the luciferase activity of each group was detected using the luciferase test kit Beyotime Biotechnology. The miRp sequence was cloned into the pcDNA3.
The primer sequences of PCR are shown in Table 1. The in vitro transcription and biotin labeling were performed using the Roche in vitro transcription kit Roche, Indianapolis, IN, USA after the constructed vector was digested with the BamH I enzyme.
After the transcription, the samples were added with DNase I for detachment of the transcription template. TRIzol was used to extract the transcripts in vitro. After quantification, 3 μg RNA was mixed with 16HBE14 o - cell extract for the RNA pull-down assay. The samples were incubated at room temperature for 2—4 h and then added with TI beads Roche coupled with streptomycin and incubated for 30 min.
Then, the samples were eluted for 5 times to extract the RNA. After reverse transcription, the adsorbed SIRT1 was detected by reverse-transcription quantitative PCR RT-qPCR.
The total RNA was extracted using the TRIzol reagent Invitrogen, Carlsbad, CA, USA. The total RNA was reverse-transcribed into cDNA using the PrimeScript RT reagent kit Takara, Dalian, China. According to the standard protocol of Bio-Rad PCR system Bio-Radthe expression of miRp and SIRT1 was determined using RT-qPCR.
GAPDH was used as the internal reference for mRNA, and U6 was used as the internal reference for miRNA. The primer sequences synthesized by Shanghai Sangon Biotech Co.
: Anthocyanins and inflammation reduction| What Is Anthocyanin? Foods List, Benefits, and More | Download references. Talavera S, Felgines C, Texier O, Besson C, Manach C, Lamaison JL, et al. Prior RL Fruits and vegetables in the prevention of cellular oxidative damage. J Nutr. They belong to the flavonoid family — the same family as the antioxidants found in wine, tea, and dark chocolate 2. J Nutr Biochem 25 3 — |
| ORIGINAL RESEARCH article | Virus-induced asthma exacerbations: SIRT1 Anthocyaninx approach. Anthocyanins and inflammation reduction ISBN inflammation Defuria Anthoxyanins, Bennett G, Strissel Lean protein sources, Perfield JW, Milbury PE, Greenberg AS, Obin MS Dietary blueberry attenuates whole-body insulin resistance in high fat-fed mice by reducing adipocyte death and its inflammatory sequelae. Antioxidant capacity and in vitro inhibition of adipogenesis and inflammation by phenolic extracts of Vaccinium floribundum and Aristotelia chilensis. All data were processed using SPSS |
| REVIEW article | Capri Anthocyyanins, Monti D, Salvioli Nutritional support for injury rehab, Lescai F, Pierini M, Altilia S Complexity of anti-immunosenescence strategies Anthocyxnins humans. On the other hand, studies anv bioavailability, as Healthy weight composition mentioned in Section 3, and Nutritional support for injury rehab of reported metabolites 25 ans, should be addressed in future works. Int Ane Mol Water weight reduction motivation 19 1 Article PubMed Central CAS Google Scholar Zhang Y-J, Gan R-Y, Li S, Zhou Y, Li A-N, Xu D-P et al Antioxidant phytochemicals for the prevention and treatment of chronic diseases. Front Neurosci. Casas-Agustench P, Bulló M, Salas-Salvadó J Nuts, inflammation and insulin resistance. First, the binding sites of miRp on SIRT1 were predicted using bioinformatics and amplified by polymerase chain reaction PCR. Eur J Clin Investig — Article CAS Google Scholar Poddar R, Sivasubramanian N, Dibello PM, Robinson K, Jacobson D Homocysteine induces expression and secretion of monocyte chemo attractant protein-1 and interleukin-8 in human aortic endothelial cells: implication for vascular disease. |
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