| BackgroundOsteoporosis(OP)is a disorder of bone metabolism,and with the development of an aging population,the prevalence of OP has increased significantly,and its serious consequences of concurrent fractures pose a serious threat to the health and quality of life of postmenopausal women.OP has become a global public health problem.According to the theory of Chinese medicine,the kidneys store essence,which is the main source of bone and marrow production.If the kidney essence is sufficient,the bone marrow will have a source of production,and the bones will be nourished and strong;if the kidney essence is deficient,the bone marrow will lack a source of production,and the bones will lose their nourishment and become impotent and weak,so the kidney should be tonified and filled with essence.Zuo Gui Wan is a famous formula in Chinese medicine for nourishing the kidneys,from Jing Yue Quan Shu.Hongqu has the ability to dry the stomach,eliminate food and lower turbidity,invigorate blood and disperse injuries,and experiments have confirmed that Hongqu can reduce osteoclast differentiation and promote bone formation.In this study,we added Hongqu to the original formula of Zuo Gui Wan,that is,Zuo Gui Wan plus Hongqu Formula.Pre-experiments in this subject confirmed that the effect of Zuo Gui Wan plus Hongqu Formula to reduce bone loss in ovariectomized rats was better than that of Zuo Gui Wan,but the mechanism is not completely clear yet.The relevance of intestinal flora,short chain fatty acids(SCFAs)and bone metabolism has been continuously demonstrated with the introduction of the concept of "gut-bone" axis.From the enzymatic digestion of carbohydrates in the intestine,SCFAs regulate the p38MAPK pathway to inhibit osteoclast(OC)differentiation;SCFAs bind to GPR41,a specific receptor on the surface of OCs,inhibit the p38MAPKNFATc1 osteoclast differentiation pathway and inhibit the differentiation of precursor cells into mature OCs.therefore,we chose the classical OC-related pathway:SCFAsGPR41-p38MAPK as the pathway in this study.Therefore,we made the following scientific hypothesis:Does Zuo Gui Wan plus Hongqu Formula regulate the SCFAsGPR41-p38MAPK pathway to exert anti-osteoporotic effect?Part1.Therapeutic effect of Zuo Gui Wan plus Hongqu Formula on Osteoporosis and its effect on SCFAs-GPR41-p38MAPK signal Pathway in Ovariectomized RatsMethod1.OP rat models were prepared,and SD female rats were divided into negative control group(NC),sham-operated group(SHAM),ovariectomy group(OVX),estradiol valerate group(EV,0.018 mg/mL),Zuogui Wan(ZGW,0.945 g of raw drug/mL),and Zuo Gui Wan plus Hongqu Formula group(ZH,1.08 g of raw drug/mL).Vaginal smears,weighing of rats and wet weight of uterus were observed to assess the modeling condition.Detection of bone mineral density(BMD),bone histomorphometric indexes by Micro-CT.Observation of bone histopathological changes(HE staining and TRAP staining)in rats.2.serum TRAP,BALP and BGP levels(ELISA assay),bone resorption-related indexes OPG,RANKL,CTSK,TRAP mRNA(qRT-PCR assay)and c-FOS,NFATc1,CTSK and TRAP proteins(WB and IHC assays).3.The changes of rat fecal microbial diversity were detected by 16S rRNA gene sequencing technique.The changes of intestinal flora diversity were observed by OTU analysis graph,Alpha and Beta diversity analysis of intestinal flora in each group of rats;the changes of relative abundance of intestinal flora in each group of rats and the difference flora between groups were analyzed at phylum level and genus level to observe the effects of Zuo Gui Wan plus Hongqu formula on the diversity and abundance of intestinal flora in ovariectomized rats.4.Gas chromatography(GC-MS)was used to detect the total amount of SCFAs and the contents of acetic acid,propionic acid,butyric acid,isobutyric acid,valeric acid,isovaleric acid and hexanoic acid in the peripheral serum of each group of rats,combined with PLS-DA analysis,univariate analysis and support vector machine analysis to observe the effects of Zuo Gui Wan plus Hongqu Formula on SCFAs and differential metabolites in the peripheral serum of ovariectomized rats.5.The expression of p38 mRNA in rat bone tissue was detected by qRT-PCR;p38 protein in rat bone tissue was detected by IHC and GPR41 and P-p38 protein in rat bone tissue were detected by WB method to explore the effect of Zuo Gui Wan plus Hongqu Formula on SCFAs-GPR41-p38MAPK signaling pathway in ovariectomized rats.Results1.General performance of rats.After 12 weeks of modeling,the vaginal smear of sham-operated rats showed a regular cycle of pre-emotional-emotional-late-emotionalinter-emotional period.The vaginal smear of the ovariectomized rats showed a continuous interphase feature and the regular erotic cycle disappeared.Compared with the SHAM group,the body weight of rats in the OVX group increased significantly(p<0.01);compared with the OVX group,the body weight of rats in the EV,ZGW and ZH groups decreased significantly(p<0.01).Compared with the SHAM group,the uterine coefficients of rats in the OVX group were significantly lower(p<0.01);compared with the OVX group,the uterine coefficients of rats in the EV,ZGW and ZH groups were not statistically different(p>0.05).This shows that the modeling was successful in this experiment.Compared with the OVX group,BMD and BV/TV were significantly increased in the EV,ZGW and ZH groups(p<0.01);Tb.N was significantly increased and Tb.Sp was significantly decreased(p<0.01;p<0.05).HE staining showed that The bone trabecular structure of bone tissue in the OVX group was sparsely arranged,disorganized and poorly connected,and TRAP staining showed more and obvious distribution of burgundy staining;the broken bone microstructure of rats in the EV,ZGW and ZH groups was improved.2.The expression of serum bone turnover markers and bone resorption-related indexes in rats.ELISA results showed that TRAP,BALP and BGP levels were significantly lower in the ZH group compared with the OVX group(p<0.01;p<0.05).qRT-PCR results showed that OPG mRNA expression was significantly upregulated in the EV and ZH groups compared with the OVX group(p<0.01),and RANKL,CTSK,TRAP and RANKL/OPG mRNA expression was significantly down-regulated in the EV and ZH groups compared with the OVX group(p<0.01;p<0.05).IHC assays showed that c-FOS,CTSK,TRAP protein expression was significantly down-regulated in the EV and ZH groups compared with the OVX group(p<0.01;p<0.05).WB assays showed that c-FOS,NFATc1,CTSK,TRAP protein expression was significantly downregulated in the EV and ZH groups compared with the OVX group(p<0.01;p<0.05).,CTSK,and TRAP protein expression were significantly down-regulated(p<0.01;p<0.05).3.Changes in the diversity of intestinal flora of rats in each group.From the OTU analysis of intestinal flora,Alpha and Beta diversity analysis,it can be seen that significant changes in intestinal flora diversity occurred in rats after ovariectomy,taking the changes in intestinal flora at the phylum level and genus level as an example,Zuo Gui Wan plus Hongqu Formula reversed the phylum Bacteroides,Firmicutes,Gemmatimonadota,Acidobacteriota and Myxococcota,and genus Muribaculaceae,Prevotella,Prevotellaceae_Ga6A1_group,UCG-005,and Sphingomonas.From the changes in the relative abundance of intestinal flora in each group of rats,it is clear that Bacteroides and Firmicutes are the dominant flora at the phylum level,and Prevotella and Bacteroidetes are the dominant flora at the genus level,and their relative abundance changed in each group.4.Changes in the content of SCFAs,a metabolite of the intestinal flora of rats in each group.Compared with the OVX group,the total serum SCFAs of rats in the EV and ZH groups were significantly higher(p<0.01),the levels of valeric acid,isovaleric acid and hexanoic acid were significantly higher in the EV and ZH groups(p<0.01;p<0.05),and the rest of acetic acid,propionic acid,butyric acid and isobutyric acid showed the same expression.Combining the results of PLS-DA analysis,univariate analysis and support vector machine analysis,hexanoic acid and isovaleric acid may be the key metabolites in the anti-osteoporosis effect of the SCFAs-mediated Zuo Gui Wan plus Hongqu formula.5.Expression of key factors of SCFAs-GPR41-p38MAPK signaling pathway.ZH activates the SCFAs-GPR41-p38MAPK signaling pathway and significantly downregulates the expression of p38 genes and proteins in the signaling pathway(p<0.01);it increases GPR41 protein expression and decreases phosphorylated p3 8 protein expression(p<0.05).Part2 Effects of Zuo Gui Wan plus Hongqu Formula serum and short chain fatty acids on osteoclasts and SCFAs-GPR41-p38MAPK signaling pathwayExperiment 1,Screening of serum concentration of Zuo Gui Wan plus Hongqu FormulaMethod1.Preparation of serum:divided into negative control serum group(Serum-NC),ovariectomy serum group(Serum-OVX),and Zuo Gui Wan plus Hongqu Formula serum group(Serum-ZH).2,The mouse mononuclear macrophage cell line RAW264.7 cells were induced to OC by RANKL,and identified by TRAP staining.the CCK-8 method was used to screen the safe dosing range of Zuo Gui Wan plus Hongqu Formula serum.3.TRAP staining was used to observe and calculate the number of TRAP+osteoclasts(≥3 nuclei)in each group;toluidine blue staining of bone resorption traps was performed on bone slices to calculate the area of bone resorption traps and to determine the optimal dosing concentration of Zuo Gui Wan plus Hongqu Formula serum.Results1.OC was successfully induced.After the addition of RANKL,the RAW264.7 cells were observed to fuse and converge gradually,and their volume increased with the appearance of vesicular structures of different sizes.After TRAP staining,cell with purplish red color and multinucleate morphology(≥3 nuclei)were identified as mature OC.2.Effects of different concentrations of Zuo Gui Wan plus Hongqu Formula serum on the proliferative activity of OC.By using CCK-8 detection method,it was determined that Zuo Gui Wan plus Hongqu Formula serum in the concentration range of 0%to 5%had no toxicity on RAW264.7 cells,so it was safe to use.3.Effect of different concentration of Zuo Gui Wan plus Hongqu Formula serum on OC differentiation.The results of TRAP staining showed that compared with OVX serum group,the number of TRAP+ osteoclasts in the groups with different concentrations(2.5%,5%)of Zuo Gui Wan plus Hongqu Formula serum group decreased to varying degrees(P<0.01),but the number of TRAP+ osteoclasts in the groups with 5%concentration of Zuo Gui Wan plus Hongqu Formula serum group decreased more significantly(P<0.01).4.Effects of different concentrations of Zuo Gui Wan plus Hongqu Formula serum on bone resorption function of osteoclasts.Compared with OVX serum,the bone resorption lacunae area in different concentration(2.5%,5%)Zuo Gui Wan plus Hongqu Formula serum group was significantly reduced(P<0.01),but the bone resorption lacunae area in 5%Zuo Gui Wan plus Hongqu Formula serum was more significantly reduced.Experiment 2,Effect of Zuo Gui Wan plus Hongqu Formula serum and short chain fatty acid on osteoclastsMethod1.SCFAs were prepared by in vitro simulation according to the content and proportion of SCFAs in peripheral blood of different groups in in vivo experiments,corresponding to the concentration of peripheral blood(1×)and bone marrow(10×).experimental groupings were as follows:1×SCFAs-NC,1×SCFAs-OVX,1×SCFAsZH,10×SCFAs-NC,10×SCFAs-OVX,10×SCFAs-ZH.OVX,10×SCFAs-ZH.Rat sera were grouped as follows:Serum-NC,Serum-OVX,Serum-ZH.2.The mRNA and protein expressions of key factors GPR41,p38 and OC specific markers were detected by qRT-PCR and WB.Results1.Effects of serum and different concentrations of SCFAs on OC differentiation and bone resorption function in various groups of rats1.1 Effects of serum and peripheral blood concentrations of SCFAs on OC differentiation and bone resorption function in rats of corresponding groupsCompared with the Serum-OVX group,the Serum-ZH group significantly reduced the number of TRAP+osteoclasts and bone resorption trap area(p<0.01),while the 1×SCFAs-OVX group was less effective in reducing the number of TRAP+osteoclasts and bone resorption trap area than the Serum-OVX group(p<0.01);compared with the Serum-ZH group,the 1 × SCFAs-ZH group was less effective than Serum-ZH group in reducing the number of TRAP+osteoclasts and the area of bone resorption traps(p<0.01).1.2 Effects of peripheral blood concentration of SCFAs and bone marrow concentration of SCFAs on OC differentiation and bone resorption function in the corresponding groupsCompared with the 1×SCFAs-OVX group,the 1× SCFAs-ZH group significantly reduced the number of TRAP+osteoclasts and bone resorption trap area(p<0.01),and the 10×SCFAs-OVX group reduced the number of TRAP+osteoclasts(p<0.01)and bone resorption trap area(p<0.05)to a more significant extent;compared with the 1×SCFAs-ZH group,the 10×SCFAs-ZH group reduced the number of TRAP+osteoclasts and the area of bone resorption traps(p<0.05)more effectively.2.Effects of serum and different concentrations of SCFAs on the expression of OC-related genes in various groups of rats2.1 Effects of serum and peripheral blood concentrations of SCFAs on NFATc1,CTSK and TRAP mRNA expression in osteoblasts of rats of corresponding groupsCompared with the Serum-OVX group,NFATc1,CTSK,TRAP mRNA expression was significantly down-regulated in the Serum-ZH group(p<0.01;p<0.05),while NFATc1,CTSK,TRAP mRNA expression was significantly up-regulated in the 1×SCFAs-OVX group(p<0.01).Compared with the Serum-ZH group,NFATc1,CTSK,TRAP mRNA expression was significantly upregulated in the 1×SCFAs-ZH group(p<0.01).2.2 Effects of peripheral blood concentration of SCFAs and bone marrow concentration of SCFAs on NFATc1,CTSK and TRAP mRNA expression in osteoblasts of corresponding groupsCompared with the 1×SCFAs-OVX group,NFATc1,CTSK,and TRAP mRNA expression were significantly downregulated in the 1×SCFAs-ZH group(p<0.01),and NFATc1,CTSK,and TRAP mRNA expression were also significantly downregulated in the 10×SCFAs-OVX group(p<0.01;p<0.05);compared with the 1×SCFAs-ZH group Compared with the 1×SCFAs-ZH group,the 10×SCFAs-ZH group significantly downregulated NFATc1,CTSK,and TRAP mRNA expression(p<0.01).3.Effects of serum and different concentrations of SCFAs on the expression of OC-related proteins in various groups of rats3.1 Effects of serum and peripheral blood concentrations of SCFAs on c-FOS,NFATc1,CTSK and TRAP protein expression in osteoblasts of rats of corresponding groupsCompared with Serum-OVX group,c-FOS,NFATc1,CTSK,TRAP protein expression was significantly down-regulated in Serum-ZH group(p<0.01;p<0.05);cFOS,NFATc1,CTSK,TRAP protein expression was significantly up-regulated in 1×SCFAs-OVX group(p<0.01).Compared with Serum-ZH group,c-FOS,NFATc1,CTSK,TRAP protein expression was significantly up-regulated in 1×SCFAs-ZH group(p<0.01).3.2 Effects of peripheral blood concentration of SCFAs and bone marrow concentration of SCFAs on c-FOS,NFATc1,CTSK and TRAP protein expression in osteoblasts of corresponding groupsCompared with the 1×SCFAs-OVX group,c-FOS,NFATc1,CTSK,TRAP protein expression was significantly down-regulated in the 1×SCFAs-ZH group(p<0.01);cFOS,NFATc1,CTSK,TRAP protein expression was significantly down-regulated in the 10×SCFAs-OVX group(p<0.01;p<0.05).Compared with 1×SCFAs-ZH group,cFOS,NFATc1,CTSK,TRAP protein expression was significantly down-regulated in 10×SCFAs-ZH group(p<0.01;p<0.05).4.Effects of serum and different concentrations of SCFAs on gene and protein expression of key factors of the pathway in various groups of rats4.1 Effects of serum and peripheral blood concentrations of SCFAs on the expression of p38 mRNA and GPR41 and P-p38 proteins,key factors of the pathway,in corresponding groups of ratsCompared with the Serum-OVX group,p38 mRNA expression was significantly down-regulated in the Serum-ZH group(p<0.01),while p38 mRNA expression was significantly up-regulated in the 1×SCFAs-OVX group(p<0.01).Compared with the Serum-ZH group,p38 mRNA expression was significantly upregulated in the 1×SCFAs-ZH group(p<0.01).Compared with Serum-OVX group,GPR41 protein expression was significantly up-regulated(p<0.01)and P-p38 protein expression was significantly down-regulated(p<0.01)in Serum-ZH group;GPR41 protein expression was significantly downregulated(p<0.01)and P-p38 protein expression was significantly up-regulated(p<0.01)in 1×SCFAs-OVX group.Compared with the Serum-ZH group,GPR41 protein expression was significantly down-regulated(p<0.05)and P-p38 protein expression was significantly up-regulated(p<0.01)in the 1×SCFAs-ZH group.4.2 Effects of peripheral blood concentration of SCFAs and bone marrow concentration of SCFAs on the expression of p38mRNA and GPR41 and P-p38 proteins,key factors of the pathway,in the corresponding groupsCompared with the 1× SCFAs-OVX group,p38 mRNA expression was significantly down-regulated in the 1×SCFAs-ZH group(p<0.01),and p38 mRNA expression was also significantly down-regulated in the 10×SCFAs-OVX group(p<0.01);compared with the 1×SCFAs-ZH group,p38 mRNA expression was significantly down-regulated in the 10×SCFAs-ZH group(p<0.01).Compared with the 1×SCFAs-OVX group,GPR41 protein expression was significantly up-regulated(p<0.05)and P-p38 protein expression was significantly down-regulated(p<0.01)in the 1×SCFAs-ZH group;GPR41 protein expression was significantly up-regulated(p<0.01)and P-p38 protein expression was significantly down-regulated(p<0.05)in the 10×SCFAs-OVX group.Compared with the 1×SCFAsZH group,GPR41 protein expression was significantly up-regulated(p<0.05)and Pp38 protein expression was significantly down-regulated(p<0.05)in the 10×SCFAsZH group.Conclusions1.Zuo Gui Wan plus Hongqu Formula can effectively improve OP symptoms,and the effect is related to the improvement of intestinal flora structure,the increase of SCFAs content,and the upregulation of key factors of SCFAs-GPR41-p38MAPK signaling pathway.2.The changes in SCFAs caused by the intervention of Zuo Gui Wan plus Hongqu Formula can effectively inhibit osteoclast differentiation,and the inhibitory effect is part of the anti-osteoporotic effect of Zuo Gui Wan plus Hongqu Formula.To sum up,the regulatory effect of Zuo Gui Wan plus Hongqu Formula on SCFAsGPR41-p38MAPK signal pathway is one of the mechanisms for the treatment of osteoporosis in ovariectomized rats,which verifies the key mediating effect of"intestine-bone" axis. |
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