| Objective1.To preliminarily explore the relationship between intestinal flora and bone metabolic diseases and screen target bacteria for the prevention and treatment of osteoporosis by studying the distribution of intestinal flora and the release of inflammatory factors in patients with postmenopausal osteoporosis2.The therapeutic principle of "tonifying kidney,invigorating spleen and invigorating the circulation of blood" was used to evaluate the efficacy of electroacupuncture in the prevention and treatment of postmenopausal osteoporosis for further demonstration the new target by preliminarily exploring the intestinal flora and bone metabolism that may be related to acupuncture3.Animal experiments were conducted to confirm the relationship between postmenopausal osteoporotic fracture and intestinal microflora disorder,and further reveal that electroacupuncture can improve postmenopausal osteoporosis,the difference in the quantity and distribution of intestinal microflora and fracture healing for providing theoretical basis for clinical prevention and treatment of postmenopausal osteoporosis.Methods1.Clinical research1.1 The intestinal colony distribution and bone metabolic factor expression in menopausal osteoporosis patients:A total of 25 postmenopausal women aged>49 years and above were recruited from June 2020 to January 2021,including 15 patients diagnosed with postmenopausal osteoporosis according to clinical Guidelines for the Diagnosis and Treatment of Osteoporosis of China,and 10 patients diagnosed without osteoprosis.Both of them were directly tested.Study factors:① Biochemical markers of bone turnover serum PINP(N-terminal propeptide of type I procollagen)and β-CTX(βCterminal telopeptide of type I collagen)were tested by Elisa.② Fecal microbes were collected for 16S rRNA sequencing to analyze the relative abundance and species differences of intestinal microbes in each group.③ Serum TNF-α,IFN-γ,IL-1β and RANKL were detected by Elisa.④ Statistical analysis was conducted to compare the relative abundance and species differences of serum bone metabolic factors and intestinal microbe between in menopausal osteoporosis group and menopausal without osteoporosis group1.2 Acupuncture bone metabolism in patients with osteoporosis and the intestinal flora of regulation:Refer to the diagnostic of deficiency of kidney and blood stasis syndrome from Guidelines for TCM Diagnosis and Treatment of Postmenopausal Osteoporosis(Bone impotence)(2019 edition),and it conforms to the western medicine diagnosis of primary osteoporosis:Most of them have a history of trauma or chronic illness,Pain in the lower back and whole body,pain with fixed place,the painful area cannot bear to press;muscle contracture,fracture,tongue with purple dark color and petechiae or bruise,tart or string pulse,assigned three groups according to willingness to treat,18 patients treated with oral calcium D;12 patients treated with oral calcium D+capsule;15 patients treated with oral calcium D+electric acupuncture.All patients were treated for 6 weeks.The following index were evaluated at week 2 and 6:① VAS score was recorded;② Serum PINP and β-CTX were tested by Elisa.③ Intestinal flora examination:Fresh feces were sequenced by 16S rRNA to analyze the relative abundance and species differences of intestinal microorganisms in each group.④Inflammatory factors:serum TNF-α,IFN-γ,IL-1β and RANKL were detected by Elisa.⑤Statistical analysis for intestinal microflora and bone metabolic factors after electroacupuncture treatment2.Animal studiesTwenty female SD rats with body weight(250-300)g were selected and randomly divided into normal group,model group,Chinese medicine treatment group,electroacupuncture treatment group,with 5 rats in each group.Establishment of the model group:postmenopausal osteoporosis was established by ovarian excision,and the rat model of osteoporosis fracture was further established after 8 weeks of routine culture.Each group received corresponding treatment for 6 weeks.Study indicators:① Bone turnover indicators such as PINP and β-CTX were measured by ELisa after 6 weeks of treatment;②After 6 weeks of treatment,the fresh feces of each group were sequenced by 16S rRNA,and the relative abundance and species differences of intestinal flora in each group were analyzed;③Serum TNF-α,IFN-γ,IL-1β and RANKL were detected by Elisa after 6 weeks of treatment.④ The expression of OPG and OPN was detected by fluorescence immunohistochemistry and Western-blot.⑤ The gut microbiome,bone metabolic factors and protein expressions of electroacupuncture with significant differences were statistically analyze.Results1.Clinical Research1.1 Comparing to postmenopausal patients without osteoporosis,serum β-CTX,RANKL,IFN-y and TNF-α were significantly higher in postmenopausal osteoporosis patients(P<0.05).There were no significant differences in IL-1β and PINP between the two groups(P>0.05).According to 16S rRNA high-throughput sequencing results,based on Shannon,Ace,Simpson and Chaos indices,the relative abundance and diversity of gut microbiome species in postmenopausal without osteoporosis patients was higher than that in postmenopausal osteoporosis patients,and the difference was statistically significant(P<0.05).The differences in gut bacteria species between the two groups were mainly concentrated in bacteroidetes,desulphurvibrios,their concomitants and their subordinate intestinal flora,and the relative abundance of Christensenellales and norank_f_norank_o_Clostridia_UCG-014 in PMOP patients were decreasing,with statistical significance(P<0.05).1.2 Comparing baseline VAS score was significantly(P<0.05)decreased treated with electroacupuncture group after treatment for 2 weeks in postmenopausal patients with osteoporosis.Meanwhile serum PINP and IL-1β increased significantly in all three groups with the decline of TNF-α(P<0.05)after treatment for 2 weeks;and serum βCTX、RANKL and IFN-γ decreased significantly in electroacupuncture group and Xianling Gubao groups after treatment for 2 weeks(P<0.05).Compared with control group,there were statistically significant differences in serum β-CTX、RANKL and IFN-γ in electroacupuncture group at 2 weeks(P<0.05).According to the gut microbiome sequencing results showed that the relative abundance of intestinal microbial species in the electroacupuncture group was the most significant at 2 weeks of treatment(P<0.05)based on Shannon,Ace and Chao indices,and the different species were mainly concentrated in the propotion of bacteroidetes and proteobacteria,and the relative abundance of faecalibacterium and Christensenellaceae probiotics in flora increased.(P<0.05)2.Animal trial studiesCompared with Calcitchi D group,serum β-CTX,RANKL and IFN-y were decreased in electroacupuncture group and Xianling Gubao group with increasing of serum PINP,there were statistical significance(P<0.05).In terms of the protein expression of OPG and OPN in femur tissue,western-blot and fluorescence immunohistochemistry showed that the protein expression of electroacupuncture group and Xianling Gubao group increased.According to the gut microbiome sequencing results,compared with the other three groups based on Sobs index,the species relative abundance of gut microorganisms in the electroacupuncture group was significantly different(P<0.05),but 1DA-Score analysis showed that the gut microorganisms with significant differences in the electroacupuncture group were mainly concentrated in the probiotics such as Bifidobacteria and Clostridium tenderness.There was statistical significance(P<0.05).Conclusion1.Serum PINP decreased and β-CTX,RANKL,IFN-γ and TNF-α increased in postmenopausal osteoporosis patients.The relative abundance of gut microbiome was lower,mainly in bacteroides,desulphurvibrios and their congeners,and the relative abundance of Christensenellales and norank_f_norank_o_Clostridia_UCG-014 in PMOP patients were decreasing,with statistical significance.2.Electroacupuncture could reduce the pain of osteoporosis,down-regulate the levels of bone metabolic factors such as β-CTX,RANKL and IFN-y,and up-regulate the relative abundance of gut microbiome and change the propotion of bacteroidetes and proteobacteria,increase the relative abundance of faecalibacterium and Christensenellaceae probiotics in flora after treatment for 2 weeks.3.In animal experiments,electric needles upregulate serum PINP levels,reduce βCTX,RANKL and IFN-γ content,and increase the protein expression of OPG and OPN in injured femur tissue to treat with the fracture,even without the relative abundance of species,but increase the proportion of probiotics such as Bifidobacteria and Pretender. |
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