Video
One Supplement PROVEN to Improve your Sleep (Not Melatonin)! [Study 62]Forskolin and scientific research -
Cells were re-suspended in 3—5 volumes of RIPA buffer and incubated on ice for 1 h. Later, samples were spun-down at 18, × g in a table top centrifuge for 15 min at 4°C. Supernatant SDS total extract was recovered in order to determine proteins concentration using Bradford Method and to prepare samples for immunoblotting adding Laemmli buffer 4× and boiling.
Cells were harvested and washed twice with cold 1× PBS and lysed in Triton extraction buffer [TEB: PBS containing 0. After a brief centrifugation at rpm at 4°C, the supernatant was removed and the pellet was washed in half the volume of TEB and centrifuged as before.
The pellet was suspended in 0. Next, the samples were centrifuged at rpm for 10 min at 4°C, the supernatant was removed, and protein content was determined using the Bradford assay as described above. Twenty to forty micrograms of proteins from whole extracts were loaded in a polyacrylamide gel Bio Rad Laboratories, Hercules, CA, United States , separated by SDS—PAGE, and transferred on nitrocellulose membrane Sigma-Aldrich, St.
Louis, MO, United States using Mini Trans-Blot BioRad Bio Rad Laboratories, Hercules, CA, United States. Data were presented as the mean ± SD of biological replicates.
P -values of less than 0. In the present study, we employed the human leukemia U cell line as a well-established and widely used model system of human leukemic cells Shayo et al.
Throughout our in vitro experiments, we used 10 μM final concentration of forskolin not toxic, submaximal dose , in agreement with several studies regarding in vitro effects by forskolin, ranging from 1 up to μM Shayo et al.
Firstly, we investigated whether GSKJ4 could have an antiproliferative action also on U cells and whether forskolin could affect such possible action. To this purpose, we treated U cells with two concentrations, 1 and 10 μM, of GSKJ4, in the presence or absence of 10 μM forskolin, for 24 and 48 h.
After treatments, direct cell number counting and PI uptake cell death assays were performed Figure 1. FIGURE 1. Effects of forskolin on the sensitivity of U cells to GSKJ4.
Panels A , B : U cells were treated or not with 1 and 10 μM GSKJ4 alone or in combination with 10 μM forskolin for 24 and 48 h. Subsequently, the cell number was recorded panel A and PI cell death assay was performed panel B. Data represent the average of four independent experiments.
The means and SD are shown. Panel C : Representative qualitative data of a typical cell death experiment with 10 μM GSKJ4 alone or in combination with forskolin for 24 h are shown. Panel D : U cells were treated or not with 10 μM forskolin and 10 μM GSKJ4 alone or in combination for the indicated times.
The image shown is representative of three different experiments with similar results. In the lower part, graphs showing the densitometric intensity of the indicated bands ratio are reported.
The intensities of signals are expressed as arbitrary units. Notably, Figure 1 shows that proliferation of U cells was inhibited by GSKJ4 in dose- and time-dependent manner. Interestingly, we found that the co-treatment with forskolin clearly enhanced the anti-proliferative effects of GSKJ4 in all the combinations.
Moreover, in Figure 1 it is also observed that forskolin, according to previous findings, does not have toxic effects in U cells Shayo et al. To increase specificity and reliability of our results, we also tested whether forskolin could potentiate the antiproliferative effects of GSKJ4 also on NB-4 cells.
Interestingly, that was the case Supplementary Data, Supplementary Figure S1. As shown in Figure 1D , in agreement with previous findings, CREB protein, a major substrate of PKA, is strongly phosphorylated in response to forskolin Naviglio et al. Notably, such phosphorylation is completely prevented by pretreatment of U cells with PKA inhibitors.
To further explore the enhancement of GSKJ4-induced anti-proliferative effects by forskolin, U cells were exposed or not control to 10 μM GSKJ4 in the presence or absence of 10 μM forskolin for 24 h.
Subsequently, the distribution of U cells in the cell cycle phases was evaluated by flow cytometric analysis of PI-stained cells Figures 2A,B.
The proportion of cells with hypodiploid DNA content sub-G1 population , characteristic of cells having undergone DNA fragmentation, a biochemical hallmark of apoptosis, was also monitored.
Figures 2A,B show that U cells treated with GSKJ4 or forskolin alone do not appear differently distributed in the cell cycle phases, with only insignificant variations, compared to control untreated ones. Thereafter, cell extracts were analyzed by western blotting to examine the levels of anti-apoptotic BCL-2 and of caspase 3 and PARP proteins Figure 2C.
This was further confirmed by examining activation of the terminal caspase-3, executioner of apoptosis, and cleavage of PARP, a known target for apoptosis-associated caspase cleavage.
Moreover, the pattern of the PARP processing proceeds in parallel with that of caspase-3 cleavage Figure 2C. FIGURE 2. Subsequently, sub-G1 and Cell Cycle Phases panels A , B were evaluated by Flow Cytometry. In panel B , representative qualitative data of a typical experiment are shown.
In panel C , the levels of the indicated proteins were assessed by western blotting from 20 μg of protein in total cell extract.
Altogether, the above data strongly suggest that forskolin enhances the sensitivity of U cells to GSKJ4, mainly by inducing apoptotic cell death. Forskolin is a direct activator of the adenylate cyclase enzyme and a largely known cAMP elevating compound.
However, forskolin can affect other cellular activities Wong et al. To verify whether the above phenotypes of forskolin could be effectively attributed to the cAMP increase, we also evaluated the effects of another cAMP elevating agent on the GSKJ4-induced cytotoxicity.
To this purpose, we treated or not control U cells for 24 h with GSKJ4 10 μM in the absence or presence of 2 mM IBMX, a broad-spectrum phosphodiesterase inhibitor Figures 3A,B. After treatments, direct cell number counting and PI uptake cell death assays were performed.
In Figures 3A,B it is shown that the proliferation of U cells was not obviously affected by IBMX, when used alone. Interestingly, we found that the co-treatment with cAMP elevating agent IBMX enhanced the anti-proliferative effects of GSKJ4, at similar extent of forskolin Figures 3A,B. Similarly to forskolin, 8-Br-cAMP, which is expected to activate both PKA and Epac, potentiated the GSKJ4-induced cytotoxicity.
Overall, the above data indicate that forskolin potentiates the sensitivity of U cells to GSKJ4 via cAMP elevation and that, very likely, PKA might be involved in.
FIGURE 3. Panels A , B : U cells were treated or not for 24 h with GSKJ4 10 μM in the absence or presence of 2 mM IBMX, 25 μM 8-pCPT-Me-cAMP, 25 μM 8-Br-cAMP. Panels C , D : U cells were treated or not for 24 h with GSKJ4 10 μM in the absence or presence of 10 μM forskolin and in the absence or presence of 10 μm PKA inhibitor KT Subsequently, the cell number was recorded panels A , C and PI cell death assay was performed panels B , D.
Data represent the average of three independent experiments. To further investigate the role of PKA on the enhancement by forskolin of the GSKJ4-induced anti-proliferative effects in U cells, we used the KT compound, which is a cell-permeable, selective, and potent PKA inhibitor Davies et al.
We found that the PKA inhibitor has a minimal effect on cell proliferation by itself. Importantly, in the presence of KT inhibitor, the enhancement by forskolin of the GSKJ4-induced cytotoxicity is clearly counteracted Figures 3C,D. Similar results were obtained also with PKA inhibitor H Supplementary Data, Supplementary Figure S2.
Currently, new efficacious therapeutic approaches are needed for the treatment of AML. Combination chemotherapy, which promises higher efficacy and lower toxicity of clinical anticancer drugs, is consistently being investigated.
The natural cAMP elevating agent forskolin has been proposed as a very promising compound for possible use in cancer therapy. Interestingly, forskolin has recently been shown to increase sensitivity to conventional antitumor drugs in myeloma, colorectal, pancreatic, and triple negative breast cancer cells Cristóbal et al.
Consistently, we describe how forskolin in combinatorial treatments markedly enhances the toxicity of the epigenetic agent GSKJ4 in leukemia cells, inducing apoptotic cell death paralleled by a strong reduction of BCL-2 protein levels, via a mechanism dependent on PKA activity. Depending on the cell type and nature of death-inducing signal, controversial effects of cAMP concerning cell death and potentiation of chemotherapeutic drugs have been described.
While the above studies clearly indicate that forskolin, as well as other cAMP elevating agents, can cause cell death or potentiate its induction by other compounds, some studies provide evidence that cAMP elevation decreases chemotherapeutic-induced cell death in cancer cells Insel et al.
Strikingly, in leukemia cells, cyclic AMP elevation, that does inhibit their proliferation and enhance their differentiation, has been shown to confer drug resistance and to protect cells against DNA damaging agents-induced apoptosis via PKA-mediated inactivating phosphorylation of Ser of pro-apoptotic Bad and activating phosphorylation of Ser of the oncogene CREB Gausdal et al.
Elevation in the global level of H3K27me3 by JMJD3 and UTX demethylases inhibition might lead to the silencing of transcription factors, such as CREB, as well as of other oncogenes, relevantly involved in leukemia Mitton et al. Understanding the mechanisms by which GSKJ4 inhibits leukemia cell proliferation is an important issue and we are planning to investigate it in future studies.
So far, whatever the exact mechanism s , here we address that exogenous addition of the cAMP elevating agent forskolin enhances the antiproliferative effects of GSKJ4 epigenetic compound via PKA in U leukemia cells. Importantly, our results confirm the cytotoxic action of GSKJ4 inhibitor on leukemia cells reinforcing the evidence of GSKJ4 as a small molecule inhibitor with anticancer potential and they demonstrate that co-administration of forskolin significantly increases the anti-cancer activity of GSKJ4 in vitro.
Remarkably, forskolin is an herbal ingredient that is already on market today as a component of weight-loss dietary supplements Kanne et al. Our opinion is that our study fits in this concept and increases the molecular evidences showing how specific signaling pathways such as PKA lead to alteration of key cancer features.
MI and MC performed the flow cytometry-based assays of cell-cycle progression and cell death. MI performed the immunoblotting experiments.
MC performed direct cell number counting. LS performed the statistical analysis and helped in the preparation of the figures. AN and AS helped to design the study and to draft the manuscript. LA and SN designed and conceived the study and drafted the manuscript.
All authors read and approved the final manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. FIGURE S1 Effects of forskolin on the sensitivity of NB-4 cells to GSKJ4.
NB-4 cells were treated or not with 1 and 10 μM GSKJ4 alone or in combination with 10 μM forskolin for 24 h. Subsequently, the cell number was recorded A and PI cell death assay was performed B.
U cells were treated or not for 24 h with GSKJ4 10 μM in the absence or presence of 10 μM forskolin and in the absence or presence of 10 μm PKA inhibitor H Castelli, G. Targeting histone methyltransferase and demethylase in acute myeloid leukemia therapy.
Onco Targets Ther. doi: PubMed Abstract CrossRef Full Text Google Scholar. Copsel, S. Cristóbal, I. Hyperphosphorylation of PP2A in colorectal cancer and the potential therapeutic value showed by its forskolin-induced dephosphorylation and activation.
Acta , — Crowley, L. Measuring cell death by propidium iodide uptake and flow cytometry. Cell counts with percent differentials were run on whole blood samples using a Coulter STKS automated analyzer Coulter Inc. Frozen serum samples were sent to the Department of Physiology at East Tennessee State University to assay thyroid stimulating hormone thyrotropin , thyroxin, total thyroxin, and fasting insulin.
A chemistry profile was run on these samples using an Immulite Mark 5 HSS chemiluminescence random access immunoassay analyzer Diagnostic Products Corporation, Los Angeles, CA following standard procedures. These analyzers were calibrated daily to controls according to manufacturer recommendations and federal guidelines for clinical diagnostic laboratories.
Analysis of these blood parameters helped determine the safety effects of this nutritional supplementation formulation on general markers of clinical health status and selected hormones. A 2 groups × 4 times analysis of variance ANOVA test with repeated measures on the second factor was performed on five variables: total body weight, total body water, body composition, appetite surveys, and psychological mood state inventories.
A 2 × 2 ANOVA test with repeated measures on the second factor was performed on the diet logs and the clinical profiles for the blood samples. Type I error was controlled at 0.
Tukey least significant difference LSD post-hoc procedures were conducted when a significance level was observed. Delta values were calculated on body composition variables to further highlight significant changes that occurred during the study.
Table 2 presents four day total nutritional intake data for the CF and P groups. Table 3 presents significant blood markers obtained throughout the study.
These hematological responses were measured for the analysis of the safety of the supplement on general markers of health. A significant decrease was noted in all of the values except for total bilirubin, which significantly increased within both groups.
All values remained within normal clinical parameters. Table 4 presents body composition data obtained during each of the four testing trials, and Figure 1 presents mean changes in body composition data from Week 0 to Week No other significant differences were observed.
Table 5 presents the appetite data obtained during each of the four testing trials. There were no significant changes observed in appetite, amount of energy, or overall quality of food. The major findings of this study were: 1. CF supplementation did not adversely affect markers of health status.
However, some interesting findings were observed that warrant additional research. The following discusses the results of this study in greater detail. The subjects involved in the study recorded all food and fluid intake during Week 0 and Week 12 of the testing sessions.
Significant decreases were seen in carbohydrate, fat, and energy intake for both groups over time. No significant differences were observed between CF and P, which suggest that supplementation had no significant effect on diet.
The changes over time could reflect subject efforts to decrease food intake in an attempt to assist in the weight loss process. It is possible that more group differences could have been observed if a specific diet were implemented into the study. This would allow for the effects of supplementation to be more closely monitored.
One purpose of this study was to determine the safety effects of forskolin supplementation on general markers of health. This was measured by monitoring changes in heart rate and blood pressure taken during each testing session, and serum and whole blood samples collected during Week 0 and Week 12 of the study.
Previous research indicated that CF causes an increase in heart rate and a decrease in blood pressure [ 14 , 15 ]. However, the results of this analysis showed that supplementation had no significant effect on either variable. The blood samples collected were assayed for muscle and liver enzymes, lipid profile, electrolytes, protein status, thyroid hormones, fasting insulin, and whole blood cell counts.
Significant changes were observed from Week 0 to Week 12 in Group CF in white blood cell count, absolute lymphocyte count, absolute neutrophil count, calcium levels, ALT, and uric acid levels.
Supplementation resulted in an increase in calcium, white blood cell, absolute lymphocyte, and absolute neutrophil counts. Decreases were observed in ALT and uric acid levels.
These variables contribute to muscle, immune, liver, and protein functions, respectively, in the body. Even though these changes occurred, the values remained within normal ranges and were relatively small.
Additionally, no significant adverse events were reported during the study that could be attributed to the supplementation protocol. Therefore, these findings suggest that CF supplementation does not appear to be associated with any significant clinical side effects.
This study was also conducted with the purpose of investigating the effectiveness of CF on the management of body composition.
Body composition was analyzed by examining measures of body weight, body water, and bone density. Previous research suggests that forskolin is associated with increased lipolysis, which would increase fat and weight loss in the body [ 13 ].
The findings from the previous pilot study suggested that supplementation significantly decreases body weight and fat, and significantly increases lean body mass in free living subjects [ 5 ]. The results from this study did not replicate those findings.
In the present study, no significant changes were observed in body weight, fat content, or lean body mass. However, statistical trends were found in total scanned mass, which could suggest some alleviation in weight gain among overweight subjects.
Overall, CF had no effect on the fat free or fat mass of the female subjects used in the study. A psychometric inventory was used to analyze mood changes during each of the four testing sessions of the study.
This analysis measured potential changes in six different mood states: tension-anxiety, depression-dejection, anger-hostility, vigor-activity, fatigue-inertia, and confusion-bewilderment.
The results of this psychological inventory revealed that regular ingestion of the supplement produced no significant alteration in mood, except for the decrease in tension-anxiety over time. This decrease could be associated with the possible effects of supplementation on body weight, which is an issue that often leads to tension-anxiety.
Trends were observed in fatigue levels. Subjects taking the supplement appeared to experience less fatigue over the course of the study. Because there were no controls for physical activity in this study, only speculation can explain the changes that occurred.
It is possible that those in CF engaged in less activity with the idea that supplementation would increase weight loss. An appetite inventory was used to assess variables of appetite during each of the four testing sessions of the study.
This inventory measured changes in hunger, appetite, satisfaction from food, fullness, and overall quality of food. No significant differences were observed in appetite, hunger, energy, or overall quality of food. This shows that the eating desires for both groups were maintained.
However, the significant time effect in satisfaction from food found in CF and P suggests that both groups received less enjoyment from the food consumed.
There was also a significant decrease noted in feelings of fullness in CF from Week 0 to Week Even though appetite and hunger were not significantly affected by supplementation, it is possible that because CF received less satisfaction from food that less food was eaten.
In conclusion, this study was performed in an attempt to further investigate the effectiveness of CF on body composition and general markers of health. The findings suggest that weeks of CF supplementation in free-living subjects have limited effects on body composition and no apparent clinically significant side effects.
However, CF has the potential to positively influence the loss and management of overall body weight possibly as a means of maintaining weight after weight loss. Still, future research is needed to further explore its capabilities.
Agarwal KC, Parks RE: Forskolin: a potential antimetastic agent. Int J Cancer. Article CAS Google Scholar. Caprioli J, Sears M: Forskolin lowers intraocular pressure in rabbits, monkeys, and man. April 30, Article Google Scholar.
Burns TW, Langley PE, Terry BE, Bylund DB, Forte LR: Comparative effects of forskolin and isoproterenol on the cyclic AMP content of human adipocytes. Life Sci. Litosch I, Hudson TH, Mills I, Li SY, Fain JN: Forskolin as an activator of cyclic AMP accumulation and lipolysis in rat adipocytes.
Mol Pharmacol. CAS PubMed Google Scholar. Diterpene forskolin Coleus forskohlii, Benth. html ]. Godard MP, Johnson BA, Richmond SR: Body Composition and Hormonal Adaptations Associated with Forskolin Consumption in Overweight and Obese Men. Obesity Research.
Srivasta SK, Khatoon CS, Mehrotra SR: Pharmacognistic evaluation of coleus forskohlii. Pharmaceutical Biology. Exercise and Sport Nutrition: A Balanced Perspective for Exercise Physiologists Database. Ltd Anand, Gujrat, India. Forskolin was procured from Phytocompounds Bangalore, India.
Acetonitrile and methanol MS grade were procured from JT Baker and Rankem. Cell culture medium and all chemicals were procured from Sigma-Aldrich St.
Louis, MO, USA. FBS, collagenase and diaspase were purchased from Invitrogen Carlsbad, CA, USA. Gum acacia was purchased from Santa Cruz Biotechnology, Inc. Dallas, TX, USA. The solution of forskolin standard 1. The mixture was then centrifuged, filtered, and used for further analysis using HPLC according to our previously described method The analysis was performed on a Phenomenex ® Luna C18 column ×4.
With gradient elution of water and acetonitrile at a flow rate of 1. The column temperature was kept at 30°C, with an injection volume was Animal husbandry and all animal experimental procedures were prior approved by the Institutional Animal Ethics Committee Registration no.
The rats were acclimated for 8 days prior to surgery. The rats were maintained on standard rodent chow diet and purified water ad libitum during the experimental period.
Adult rats with a drill-hole 0. Furthermore, this model is useful for the determination of effective osteogenic dose of a drug.
Twenty four female SD rats ± 20 g were used for femur osteotomy following a previously described protocol All the treatments were given daily for 12 days. After sacrifice, bones were collected and processed for calcein labeling studies according to our previously published protocol.
Sections were photographed using a confocal microscope Leica TCS SP-8, Wetzlar, Germany and analyzed using LAS-X software. Modeling-directed bone formation is the dominant event in growing rats, which enables evaluation of the osteogenic response in intact skeleton 21 , Treatments were given for 1 month.
For dynamic histomorphometry study to measure surface-referent bone formation parameters , each animal was given two s. At the end of the experiment, bones were collected for μCT, bone strength, and histomorphometry analyses, for which the details are given below.
Femur length was measured with a Vernier caliper. To this aim, 24 SD rats ± 20 g, 3 months old underwent either bilateral OVX or a sham operation ovary intact 19 , All the therapies were given daily for 3 months.
before sacrifice at the interval of 10 days 19 , After the treatment, serum samples, and bones femur, tibia, and L5 vertebrae were dissected and stored at °C for further studies. Throughout the experimental period, the body weights of each study group were measured once a week.
Singapore 24 h before the end of the experiment Total body mass and lean mass were plotted, while fat mass was plotted by normalizing with the total body mass. Bone samples were scanned using a SkyScan computed tomography μCT scanner SkyScan, Ltd.
CTAn software was used manually to quantify various bone parameters as described previously Reconstructed μCT images underwent a blind evaluation by a third person to determine the extent of bone loss. The degree of bone loss was assessed using reconstructed μCT images that had undergone a blind examination by a third person.
Biomechanical strength was measured by L5 compression test using a bone strength tester, TK C Muromachi Kikai Co. Tokyo, Japan according to our previously published method The rat femur bone was cut from mid-diaphysis with a low-speed diamond blade saw IsoMet; Buehler, Lake Bluff, IL, USA , and after that, the samples were kept in epoxy for nearly 2 h for proper cured and, further samples were polished under the ground Buehler Eco grinder and polisher with the abrasive papers of , , and grit size under the cooling condition and polished with a diamond solution of particle sizes of 1, 0.
After completion of polishing, samples were sonicated for 10 min. The experiment was performed on the T Tribo Indenter Hysitron Inc. Eight indents with a peak load of µN were applied to cross-section of the bone. The load sequence consists a loading time of 10 s, an unloading segment, and a hold for 10 s.
The resultant load-displacement curve was used to calculate the reduced modulus Er and hardness H by the method of Oliver and Pharr 26 , From the obtained data, we calculated the following parameters: carbonate-to-phosphate ratio area ratio of the carbonate peak [ cm -1 ] to phosphate peak [ cm -1 ] , carbonate-to-amide I ratio area ratio of the carbonate peak [ cm -1 ] to the amide-1 peak [ cm -1 ] and mineral crystallinity ratio intensity ratio of [ to cm -1 ] , which is related to crystal size and stoichiometric perfection.
The amide I band peak contains several sub-peaks that provide information about the collagen matrix and the location of cross-linkage and non-cross linkage.
The sub-band of the amide I peak were fited with Gaussian curves at , , , , , and cm -1 using peak analyzing tools OriginPro 8. Rat cross-linked C-telopeptide of type I collagen CTX-1 kit Cat.
E-EL-R and pro-collagen type I N-terminal propeptide PINP kit Cat. Rat pups 1 to 2-day-old were treated with vehicle or forskolin 1- and 2. After treatment, calvaria were removed and processed for RNA isolation by trizol method qPCR was performed by SYBR green chemistry Thermo Fisher Scientific, Ealtham, MA, USA for the quantitative determination of bone morphogenetic protein 2 BMP2 , type 1 collagen Col I , receptor activator of nuclear factor kappa-B ligand RANKL , and osteoprotegerin OPG as described previously cDNA was synthesized by using 2μg RNA Cat no.
All genes were analyzed using a real-time PCR machine QuantStudio © 3 Real-time PCR Instrument, A , keeping GAPDH as control. Primer sequences are listed below:. Bone marrow was collected from rat femur by flushing out using PBS and cells were measured by a hemocytometer.
After every 48 h media was changed for 21 days. Rat pups 1 to 2-day-old were used to culture calvarial osteoblasts RCO as described previously The adherent cells were treated with CFE 7.
RCO were treated with CFE or forskolin for 0 min, 5 min, 15 min, 30 min, 60 min and 90 min. After treatments, cAMP and cGMP levels were determined by ELISA kits Cayman Co.
Data are presented as the mean ± standard error of the mean SEM. One-way ANOVA with a post hoc Tukey test using GraphPad Prism 5 and a significance level of 0. An unpaired t-test using GraphPad Prism 5 with a significance level of 0. The HPLC method was applied for simultaneous quantification of analytes in CFE.
The chromatograms for the standard mixture and samples are presented in Supplementary Figure 1. The chromatogram of the sample solution obtained in the test for the content of forskolin showed a major peak at a retention time corresponding to that of the forskolin reference standard.
Other diterpene peaks in the sample chromatogram exhibited an additional peak corresponding to isoforskolin. Forskolin and isoforskolin content in the CFE were Osteoblasts are the principal cells that are involved in fracture healing, so we firstly studied the effect of CFE on the differentiation of RCO by ALP activity.
As forskolin is an AC activator, we next studied the effect of CFE Furthermore, at the same concentration, CFE Figure 1 CFE stimulated osteoblast differentiation, cAMP and cGMP in vitro and promoted bone regeneration at the femur osteotomy site.
A RCO were treated with CFE, and differentiation was assessed by ALP assay. B RCO were treated with CFE at the indicated time points and cAMP and C cGMP production were measured. D Adult female rats were treated with vehicle and CFE at indicated doses after femur osteotomy for 12 days, and representative images 10X of calcein deposition at the osteotomy site are shown.
In obese men, mg CFE has been used to study its impact on body mass 5 , 9. Compared with vehicle-treated rats, CFE at all doses significantly increased calcein intensity Figures 1D, E. Trabecular bones at metaphysis and cortical bone parameters were studied using μCT. N and trabecular thickness Tb.
Th compared with control Figure 2B. Cortical parameters including cortical thickness Ct. Th and bone area B. Ar were significantly increased by CFE over the control Figure 2C. Figure 2 CFE promoted new bone formation in growing rats.
A Femur length. B Representative μCT images left panel and quantitative μCT parameters of the tibia metaphysis right panel.
N, trabecular number; Tb. Th, trabecular thickness. C Representative μCT images upper panel and quantitative μCT parameters of the femur and tibia diaphysis lower panel. Th, Cortical thickness and B. Ar, bone area. D Left panel showing the representative images of double calcein labeling scale bar, µm and histomorphometry parameters right panel of the indicated groups.
E 3-point bending strength of femur was determined by a bone-strength tester. vehicle-treated group. The effect of CFE on bone accrual was measured by dynamic histology by time-spaced calcein labeling study in the periosteal p region of the femur diaphysis.
Increase in the surface referent bone formation parameters indicative of increased periosteal apposition complemented our observation of higher Ct. Th in the CFE group over the control and is likely to afford greater resistance to fracture Accordingly, we measured the bending strength of femur and observed that maximum power and energy to failure were significantly higher in the CFE group compared with control Figure 2E.
The treatment of CFE had no effect on the body weight compared to the vehicle treated group data not shown.
Because CFE promoted bone regeneration and stimulated modeling-based bone growth, we speculated that it would have bone conserving effect in OVX model of osteopenia.
At the end of 3 months of treatment, body composition of all groups were assessed by Echo-MRI. Compared with the sham, OVX rats had increased total body mass, lean mass, and fat mass.
CFE had no effect on OVX-induced increase in total body mass and lean mass but significantly decreased fat mass Supplementary Figure 2. We next studied the effects of CFE on appendicular tibia and axial L5 skeletons of OVX rats Figure 3A for representative images.
N and Tb. Th were reduced in the OVX group and CFE significantly increased Tb. Th only. Consequently, trabecular spacing Tb.
sp that was increased in the OVX group was significantly reduced by CFE treatment. Th were significantly reversed by CFE with consequent recovery of Tb. Figure 3B. Figure 3 CFE prevented bone loss in osteopenic rats. A Representative images of tibia metaphysis, and L5 vertebrae are shown.
B Shown are the quantitative μCT parameters of the tibia metaphyses, and L5. BMD, bone mineral density; Tb. Sp, trabecular spacing; Conn. C The L5 compression strength was determined by a bone-strength tester.
Although there was not a significant difference among groups in maximum power and failure energy of the L5 after the end of the treatment, stiffness was significantly lower in the OVX compared with the other groups Figure 3C. We next studied whether the preservation of bone mass and strength by CFE in OVX rats occur by an osteogenic mechanism.
Complementing the osteogenic effect of CFE through increase in surface-referent bone formation, ex vivo mineralization of BM stromal cells in the CFE-treated OVX rats was significantly higher than the OVX group Figure 4B.
Figure 4 CFE has an osteoanabolic effect in osteopenic rats. A Upper panel showing representative images scale bar, µm of single and double calcein labeled bone surfaces at tibia metaphysis; white arrows- single label and yellow arrows- double label surfaces; and the lower panel showing the histomorphometry parameters in the indicated groups.
B Ex vivo mineralization assay was performed in bone marrow stromal cells obtained from the indicated groups. C Serum procollagen type I N-propeptide PINP , cross-linked C-telopeptide of type I collagen CTX1 levels and their ratio were determined by ELISA from the serum of rats with indicated treatments.
Consistent with the in vivo and ex vivo osteogenic effect of CFE, we observed that the serum bone formation marker, PINP, that was significantly decreased in the OVX group was maintained to the sham level by CFE.
Conversely, CFE suppressed the OVX-induced increase in CTX-1, the serum resorption marker. The mineral-based parameters including mineral crystallinity and carbonate:phosphate ratio were respectively decreased and increased in the OVX group, and CFE treatment maintained these parameters to the levels of sham.
The carbonate:amide-I ratio was significantly increased in the OVX group, while CFE treatment maintain this parameter to the sham level Table 1. We next studied the material properties of bones by nanoindentation. Under a µN load, the OVX group had significantly lower modulus and hardness compared with sham and CFE groups Table 1.
Since CFE is rich in forskolin we surmised that it contributes to the osteogenic effect of the extract. In the osteoblast ALP assay for assessing differentiation, the EC 50 of forskolin was 3. Prolonged increase in cAMP as caused by theophylline caused osteoblast apoptosis On the other hand, PTH through Gs α -coupled activation of PTH receptor-1 stimulates AC to increase osteoblastic cAMP which leads to osteoblast differentiation Therefore, we compared the intracellular cAMP kinetics of forskolin at 10 nM with PTH.
Forskolin had a greater total cAMP level than PTH Figure 5B. Furthermore, forskolin increased the intracellular cGMP levels in the RCO compared with vehicle treated RCO Figure 5C.
Figure 5 Forskolin has osteogenic effect in vitro and in vivo. A RCO were treated with forskolin at the indicated concentrations and differentiation was assessed by ALP assay. B RCO were treated with forskolin 10nM for the indicated time points and intracellular cAMP and C cGMP production were measured.
D Rat pups 1-day old were injected with forskolin at the indicated doses for 5 consecutive days and the relative expression of osteogenic genes in the calvarial tissue were measured.
Acute myeloid Fors,olin AML is an aggressive hematological malignancy occurring very often in rseearch adults, with poor Vegetable garnishing ideas depending Balanced caloric intake both rapid disease progression Vegetable garnishing ideas drug resistance occurrence. Therefore, new therapeutic approaches are demanded. Epigenetic marks play a relevant role in AML. GSKJ4 is a novel inhibitor of the histone demethylases JMJD3 and UTX. To note GSKJ4 has been recently shown to act as a potent small molecule inhibitor of the proliferation in many cancer cell types. Losing weight can be challenging. Those who regain their Fofskolin weight Heart health benefits more likely to seek solutions such as dietary Forskolin and scientific research and herbal medicines. Forskolin and scientific research such scientifc is forskolin, a natural plant compound Foskolin Vegetable garnishing ideas said to be an impressive weight loss supplement. But does it really work? Forskolin is an active compound found in the roots of the Indian coleus Coleus forskohliia tropical plant related to mint. For centuries, this plant has been used in traditional herbal medicine to treat various conditions and diseases 2. Weight loss supplements can allegedly promote weight loss in several ways, including by 5 :.
die Interessante Variante
Sehr, sehr
Die sehr ausgezeichnete Idee
der Unsinn welcher jenes
Wacker, Sie hat der einfach prächtige Gedanke besucht