Mechanism Of Zhuanggu Shubi Ointment In The Intervention Of Postmenopausal Osteoporosis Based On Intestinal Flora

Objective:In this study,a rat model of postmenopausal osteoporosis(PMO)was replicated by bilateral ovarian denudation.Zhuanggu shubi ointment,a clinical experience formula developed by Professor Li Qingsheng in Yunnan Province,was used for drug intervention.Changes in bone density,intestinal mucosal barrier,inflammatory response,intestinal flora and other related indicators were observed in rats,to elucidate the therapeutic efficacy mechanism of the PMO intervention by Zhuanggu shubi ointment,to provide theoretical basis for the pursuit of multi-targeted treatment of PMO in Traditional Chinese Medicine(TCM),and to provide the new ideas for clinical prevention and treatment of PMO.Methods:2.Model replication: The PMO rat model was replicated by bilateral ovary castration,and the model was evaluated by detecting bone mineral density,bone histopathological changes and related bone transformation indexes in serum.2.Drug intervention: The drug intervention group was given Zhuanggu shubi ointment decoction by intragastric administration.The ovarian castration group and sham operation group were given the same amount of sterile distilled water for 56 consecutive days.3.Indicator detection: Enzyme linked immunosorbent assay(ELISA)was used to detect the contents of lipopolysaccharide(LPS),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and secretory immunoglobulin A(SIg A)in the colon tissue of rats.The expression of tight junction proteins Zonula occluden-1(ZO-1),Occludin and Claudin 1 in the colon tissue of rats were detected by immunofluorescence.16 S r RNA gene high-throughput sequencing was used to detect intestinal flora in rat stool,gas chromatography-mass spectrometer(GC-MS)was used to detect short-chain fatty acids(SCFAs)in rat serum and stool.Results:1.Therapeutic effect of Zhuanggu shubi ointment on PMO:(1)Zhuanggu shubi ointment improved the bone mineral density of PMO rats:Compared with sham operation group,trabecular bone mineral density(Tb.BMD),trabecular bone volume fraction(Tb.BV/TV),trabecular number(Tb.N)and trabecular thickness(Tb.Th)of rats in ovarian castration group were significantly decreased(P<0.05;P<0.05;P<0.05;P<0.05),the trabecular separation(Tb.Sp)showed an increasing trend(P>0.05).Compared with ovarian castration group,Tb.BMD,Tb.BV/TV,Tb.N and Tb.Th of Zhuanggu shubi ointment group were significantly increased(P>0.05;P<0.05;P<0.05;P<0.05),Tb.Sp showed the decreasing trends(P>0.05).(2)Zhuanggu shubi ointment promoted bone formation and inhibited bone resorption in PMO rats:Compared with sham operation group,the contents of bone r-hydroxy glutamic acid protein(BGP)、procollagen type I N-terminal propeptide(PINP)in serum of ovarian castration group was significantly decreased(P<0.05;P<0.05),the contents of C-terminal telopeptide of type-Ⅰcollagen(CTX-1)and tartrate resistant acid phosphatase-5b(TRACP-5b)were significantly increased(P<0.05;P<0.05).Compared with ovarian castration group,the contents of BGP and PINP in serum of Zhuanggu shubi ointment group were significantly increased(P<0.05;P<0.05),the contents of CTX-1 and TRACP-5b were significantly decreased(P<0.05;P<0.05).(3)Zhuanggu shubi ointment improved the femoral structural injure in PMO rats:Hematoxylin eosin(HE)staining results showed that compared with the sham operation group,the bone trabecular junction break points increased,the bone trabecular thin and sparse distribution,and the number of fat cells in the bone marrow cavity increased in the ovary castration group.After the intervention of Zhuanggu shubi ointment,the above conditions were improved.Masson staining results showed that compared with the sham operation group,the collagen fibers of femur in the ovary castration group were pale blue,the collagen fiber junction breakpoints increased,and the collagen fiber distribution was sparse.After the intervention of Zhuanggu shubi ointment,the above conditions were improved.2.The mechanism of Zhuanggu shubi ointment in treating PMO:(1)Zhuanggu shubi ointment improved the colonic structural damage of PMO rats:Compared with the sham operation group,the intestinal recess of the rats in the ovarian castration group was shallower,the submucosa edema and inflammatory cell infiltration in the basal layer appeared to a certain extent,and the above conditions were improved after the intervention of Zhuanggu shubi ointment.(2)Zhuanggu shubi ointment repaired intestinal mucosal barrier injury in PMO rats:Compared with sham operation group,the expression of ZO-1,Occludin and Claudin-1 in colonic tissue of ovarian castration group significantly decreased(P<0.05;P<0.05;P<0.05),SIg A in colonic tissue was significantly decreased(P<0.05).After the intervention of Zhuanggu shubi ointment,the above indexes were significantly increased(P<0.05;P<0.05;P<0.05;P<0.05;P<0.05).(3)Zhuanggu Shubi Ointment reduced the intestinal inflammation of PMO rats:Compared with the sham operation group,the contents of TNF-α,IL-6 and IL-1βin the colonic tissue of rats in the ovarian castration group were significantly increased(P<0.05;P<0.05;P<0.05),LPS showed an increasing trend(P>0.05).Compared with ovarian castration group,the contents of TNF-α、IL-1β、IL-6 and LPS in colonic tissue of rats in Zhuanggu shubi ointment group were significantly decreased(P<0.05;P<0.05;P<0.05;P<0.05).(4)Zhuanggu shubi ointment regulated the changes of intestinal flora in PMO rats:Compared with sham operation group,the Chao 1,Simpson and Shannon indexes of rats in ovarian castration group showed the increasing trends(P>0.05;P>0.05;P>0.05).Compared with ovarian castration group,the Chao 1,Simpson and Shannon indexes of Zhuanggu shubi ointment group showed the decreasd trends(P>0.05;P>0.05;P>0.05).The bacterial community structure of each group was separated.Compared with the sham operation group,the relative abundance of Bacteroidetes and Firmicutes in the ovarian castration group showed the increased trends(P>0.05;P>0.05),Verrucomicrobiota and Euryarchaeota showed the decreased trends(P>0.05;P>0.05).Compared with the ovarian castration group,the relative abundance of Bacteroidetes,Firmicutes and Verrucomicrobiota in the Zhuanggu shubi ointment showed the decreased trends(P>0.05;P>0.05;P>0.05),Euryarchaeota showed an increased trend(P>0.05).After the intervention of Zhuanggu shubi ointment,Proteobacteria and Actinobacteriota were significantly increased(P<0.05;P<0.05),Campilobacterota was significantly decreased(P<0.05).Compared with the sham operation group,the relative abundance of Akkermansia、Lactobacillus、Prevotellaceae＿UCG-003 and Methanobrevibacter in the ovarian castration group showed the decreased trends(P>0.05;P>0.05;P>0.05;P>0.05).Compared with the ovarian castration group,the relative abundance of Akkermansia 、 Lactobacillus and Prevotellaceae＿UCG-003 in the Zhuanggu shubi ointment showed the decreased trends(P>0.05;P>0.05;P>0.05),Muribaculaceae and Methanobrevibacter showed the increased trends(P>0.05;P>0.05).After the intervention of Zhuanggu shubi ointment,Prevotella was significantly increased(P<0.05),Helicobacter was significantly decreased(P<0.05).Linear discriminant analysis effect size(LEf Se)analysis showed that there were two different groups between sham operation group and ovarian castration group,namely o＿Oscillospirales and f＿Rikenellaceae.There were seven different groups between ovarian castration group and Zhuanggu shubi ointment group.The main microflora in ovarian castration group were g＿Rikenellaceae＿RC9＿gut＿group 、o＿Oscillospirales and f＿Rikenellaceae.The main microflora in Zhuanggu shubi ointment group are g＿Muribaculaceae 、f＿Muribaculaaceae、 p＿Proteobacteria、g＿Prevotella and c＿Gammaproteobacteria.KEGG function results showed that thirteen functional pathways with statistical difference were predicted in the sham operation group VS ovarian castration group,including one down-regulated functional pathway(P341-PWY)and one upregulated functional pathway(TYRFUMCAT-PWY).A total of seventy functional pathways with statistical difference were predicted in the analysis of ovarian castration group VS Zhuanggu shubi ointment group,including thirty-one upregulated functional pathways.Statistically significant differences were found in GLYCOLYSIS-TCA-GLYOX-BYPASS、TCA-GLYOX-BYPASS and PWY-7007.(5)Zhuanggu shubi regulated the expression of SCFAs in faeces and serum of PMO ratsCompared with sham operation group,the levels of acetic、propionic、butyric、isobutyric、valerate、isovalerate acids in faeces of rats in ovarian castration group showed the decreased trends(P>0.05;P>0.05;P>0.05;P>0.05;P>0.05;P>0.05),caproic acid showed an increased trend(P>0.05).Compared with ovarian castration group,the levels of acetic、propionic、butyric、valeric acids in faeces of Zhuanggu shubi ointment group showed the increased trends(P>0.05;P>0.05;P>0.05;P>0.05),isobutyric、isovaleric、caproic acids showed the decreased trends(P>0.05;P>0.05;P>0.05).Compared with sham operation group,the levels of acetic、propionic acids in serum of rats in ovarian castration group showed the decreased trends(P>0.05;P>0.05),butyric、isobutyric、valerate、isovalerate、caproic acids showed the increased trends(P>0.05;P>0.05;P>0.05;P>0.05;P>0.05).Compared with ovarian castration group,the levels of acetic acid of Zhuanggu shubi ointment group showed an increased trend(P>0.05),propionic、butyric、isobutyric、valerate、isovalerate、caproic acids showed the decreased trends(P>0.05;P>0.05;P>0.05;P>0.05;P>0.05;P>0.05).Conclusions:1.Zhuanggu shubi ointment improved bone mineral density in PMO rats,promoted bone formation,inhibited bone resorption related indicators,and restored femoral structural damage,thus achieving the effect of treating PMO.2.The mechanism of Zhuanggu shubi ointment in the treatment of PMO was through regulating intestinal flora and SCFAs,repairing the damage of intestinal mucosal barrier,down-regulating intestinal inflammatory response.3.Intestinal differential bacterium(g＿Muribaculaceae and g＿Prevotella)might play the positive roles in the treatment of intestinal mucosal barrier injury and inflammatory reaction of Zhuanggu shubi ointment in PMO.