Clinical Effect Observation And Mechanism Study Of Acupoint Catgut Embedding On Postmenopausal Osteoporosis

Objective:PMOP is a common clinical disease that occurs in 25%-50%postmenopausal women and is the main type of primary osteoporosis.Most patients have symptoms such as general weakness,bone pain,and fracture proneness,which seriously affect patients' life quality.Acupoint catgut embedding has a definite therapeutic effect on PMOP,but the mechanism of acupoint catgut embedding for PMOP is still not clear.Studies have shown that GM can affect bone metabolism,which is closely related to the occurrence of OP.Metabonomics can measure the composition of all metabolites in biological samples and their dynamic changes under internal and external stimulation.We intend to observe the therapeutic effect of acupoint catgut embedding on PMOP,and to preliminarily explore the mechanism through metabonomics and GM detection in this study.Methods:(?)Clinical Part90 female patients aged 50-65 who have been naturally menopausal for more than 2 years and met OP through BMD examination were randomly divided into drug group and drug+embedding group,with 45 patients in each group.After removing the abscission cases,42 cases were included in the drug group and 40 cases in the drug+embedding group.In the drug group,caltrate D was given orally,2 tablets per time,once a day;alfacalcidol was given orally,0.25 ?g/time,once a day.The two drugs were taken together for 6 months.In the drug+embedding group,on the basis of oral medicine,acupoints such as Pishu(BL20),Sanyinjiao(SP6),Shenshu(BL23),Zusanli(ST36),Zhongwan(RN12),Guanyuan(RN4),etc.were selected for embedding therapy,once a week,one side of acupoint was selected each time,and a single acupoint was selected alternately.The treatment lasted for 6 months.Before treatment,after 3 and 6 months of treatment,BMD of the lumbar spine and femoral neck was tested,and serum OPG,RANKL,E2,ALT,TG,TC,Cr,and BUN levels were detected,and OP efficacy determining,VAS,OP syndrome score,spleen and stomach function assessment scale were used for evaluation.(?)Expremental PartThirty-two 3-month-old female SD rats were randomly divided into blank group A,sham operation group B,model group C and embedding group D,with 8 rats in each group.In group C and D,bilateral ovaries were removed to make PMOP model,while in group B,only a small amount of adipose tissue around ovaries was removed.The rats in group A,group B and group C did not receive any intervention.The rats in group D were selected "Shenshu"(BL23)and"Housanli"(ST36)for embedding treatment,alternately on both sides.The therapy was performed once a week for 12 consecutive weeks.After the intervention,BMD of femur and tibia,serum E2,PTHrP,OPG,RANKL,1,25(OH)2D3,IGF-1,TNF-?,IL-1,LPS,MAPK/p38 were detected respectively.The composition and abundance of faeces flora were detected based on 16s rRNA augmentation and high-throughput sequencing technology,gas chromatography-mass spectrometry(GC-MS)was used to detect the composition and content of SCFAs,nontargeted metabolomics was performed using liquid mass spectrometry(LC-MS)to detect fecal differential metabolites and related metabolic pathways.Results:(?)Clinical PartThe BMD of lumbar vertebrae and femoral neck in both groups after treatment were higher than those before treatment,and BMD increased with the extension of treatment time.Although the BMD of the drug+embedding group was significantly higher than that of the drug group,there was no statistical difference(P>0.05).After treatment,the scores of VAS and OP syndrome in both groups decreased with the prolongation of treatment time.Compared with the drug group,the score of OP syndrome in the drug+embedding group decreased more significantly(P<0.05).After 3 months and 6 months of treatment,the spleen and stomach function scores in the drug+embedding group were significantly lower than those before treatment(P<0.01),while there was no significant change in the drug group before and after treatment(P>0.05).The decrease degree of spleen and stomach function in the drug+embedding group was statistically significant compared with the drug group(P<0.01).There was a significant difference in the markedly effective rate of OP between the two groups(P<0.01).The serum E2 level of the drug+embedding group was significantly increased after treatment compared with that before treatment(P<0.01),and the E2 level of the two groups was significantly different after 6 months of treatment(P<0.05),but there was no significant difference in the increasing trend of E2 between the two groups(P>0.05).After 6 months of treatment,the OPG of the two groups was significantly higher than that before treatment(P<0.01),and the increasing trend of OPG in the drug+embedding group was better than that in the drug group(P<0.01).After treatment,the RANKL in the drug+embedding group was significantly lower than that before treatment,but there was no significant change in RANKL in the drug group before and after treatment(P>0.05).The reduction of RANKL in the drug+embedding group was better than that in the drug group(P<0.01).ALT,Cr and BUN were in the normal range before and after treatment,and there was no significant difference between the two groups(P>0.05).After 3 months and 6 months of treatment,the TC and TG in the drug+embedding group were significantly lower than those before treatment,but there was no significant change in the drug group before and after treatment(P>0.05).The effect of the drug+embedding group was better than that of the drug group in reducing TC and TG(P<0.01).(?)Experimental PartBMD of femur and tibia in model group decreased significantly,and BMD in embedding group was significantly increased compared with model group(P<0.05).E2,PTHrP,1,25(OH)2D3,OPG,OPG/RANKL and IGF-1 in the model group were significantly decreased compared with the blank group and the sham group,while those in the embedding group were increased compared with the model group.Except for IGF-1,the differences in other indexes among the groups were statistically significant(P<0.01 or P<0.05).RANKL,TNF-?,IL-1,LPS,MAPK/p38 in the model group were significantly higher than those in the blank group and the sham group,while those in the embedding group were significantly lower than those in the model group.Except for LPS,there were significant differences in other indexes among groups(P<0.01 or P<0.05).GM abundance and species composition were analyzed by Alpha diversity,and there was no statistically significant difference between each group.However,by Beta diversity analysis,there were statistically differences between the blank group and the shame operation group,the blank group and the model group,and the blank group and the embedding group(P<0.01).It can be seen in the Beta diversity index heat map,PCoA analysis,PCA analysis and UPGMA cluster tree that the species composition and abundance of GM in the blank group,the shame operation group,and the embedding group are more closer,which were quite different from those in the model group.There were some differences in the species composition of the four groups.The T-test on the different species between the groups showed that the GM that has changed in the model group mainly included mollicutes,actinobacteria,acidobacteria,chloroflexi,and fusobacteria.The abundance of probiotics in the embedding group such as lactobacillales(lactobacillaceae,lactobacillus),bacillales,and streptococcus was significantly higher than that in the model group(P<0.05).The GM which was intervened by embedding mainly included acidobacteria,chloroflexi,and fusobacteria,etc.,and there was also significant intervention effect in the different taxonomic levels of actinobacteria,firmicutes,proteobacteria,and bacteroides.Compared with blank group and sham group,SCFAs contents in model group were significantly decreased(P<0.01 or P<0.05),but there was no significant change in embedding group(P>0.05).Compared with model group,the content of SCFAs in embedding group increased to a certain extent,and the contents of acetic acid,butyrate,iso-valerate,valerate and caproic acid in embedding group were significantly different from that in model group(P<0.05).Compared with the sham operation group,the embedding group mainly changed in the following pathways,such as steroid hormone biosynthesis,cortisol synthesis and secretion,Cushing syndrome,glycolysis and gluconeogenesis,pentose phosphate pathway,and vitamin B6 metabolism,etc.(P<0.05).The content of pregnenolone,tryptophan,bilirubin and other substances in feces were significantly up-regulated,while urine cortisol,xanthuric acid,3-hydroxy-5-pregnan-20-one,3-glyceraldehyde phosphate,5-phosphate ribulose,etc.were down-regulated(P<0.05).Compared with the model group,the embedding group had significant changes in the pathways including tryptophan metabolism,steroid hormone biosynthesis,platelet activation,linoleic acid metabolism,and serotonergic synapse,etc.The content of metabolites such as arachidonic acid and prostaglandin G2 were significantly down-regulated compared with the model group(P<0.05).Conclusions:Acupoint catgut embedding can effectively improve the clinical symptoms and spleen and stomach function of patients with PMOP,mainly by increasing the levels of estrogen,1,25(OH)2D3,PTHrP and IGF-1,and reducing the levels of TNF-?,IL-1,LPS and MAPK/p38,regulating the OPG/RANKL system,which can promote bone formation and inhibit bone resorption.Acupoint catgut embedding can increase the abundance of probiotic bacteria such as lactobacillales(lactobacillaceae,lactobacillus).bacillales,and streptococcus,etc.which also indirectly increased the content of SCFAs.In addition,it can also reduce the abundance of pathogens such as mollicutes,actinobacteria,acidobacteria,chloroflexi,and fusobacteria,etc.All these may be the mechanism of embedding to prevent and treat PMOP.Tryptophan metabolism,steroid hormone biosynthesis,platelet activation,linoleic acid metabolism,serotonergic synapse and other pathways may be the main pathways for embedding to interfere with PMOP.In addition,acupoint embedding can reduce or inhibit inflammatory response by reducing the content of arachidonic acid and prostaglandin,which may be one of the mechanisms of embedding in the treatment of PMOP.