| BackgroundKnee osteoarthritis(KOA)is a degenerative cartilage disease of the bone and joint.At present,there are many studies on the pathogenesis of KOA.As an important part of human body,intestinal microbes interact with each other to maintain the stability of intestinal microecology.Once the intestinal microhomeostasis is out of balance,it will produce corresponding symptoms and even produce disease.At present,the pathogenesis of KOA has been proved to be closely related to some pathogenic bacteria,and acupuncture can effectively improve the knee joint pain and stiffness,while it has been rarely reported whether acupuncture can improve the knee joint function by regulating the number and structure of intestinal flora in patients.Objective1.Compare the degree of improvement of joint pain,stiffness and mobility in patients with KOA by electroacupuncture/sham,and compare the difference of clinical efficacy between the electroacupuncture group and the sham group.2.Compare the intestinal flora of KOA patients with that of healthy subjects,and screen the major pathogenic bacteria groups causing the onset of knee osteoarthritis.3.The differences of intestinal flora in KOA patients treated with electroacupuncture and sham acupuncture were compared to investigate the effects of electroacupuncture on intestinal flora in patients with knee osteoarthritis.4.Analyze the correlation between changes in intestinal flora before and after acupuncture treatment and clinical indicators and efficacy,and reveal the potential mechanism of acupuncture to improve intestinal flora in KOA patients.Methods1.From April 2018 to January 2019,60 patients with knee osteoarthritis were included in the study from Dongzhimen hospital of Beijing university of Chinese medicine,Dongfang hospital of Beijing university of Chinese medicine,Beijing hospital of traditional Chinese medicine affiliated to Capital medical university,Guang ’anmen hospital of Chinese academy of traditional Chinese medicine and acupuncture and moxibustion hospital of China academy of traditional Chinese medicine.They were randomly divided into EA patients(n=30)and SA patients(n=30).Both groups received treatment 3 times a week for 8 weeks.Knee pain,function and stiffness were evaluated with WOMAC before and after treatment.Differences in the effective response rates between the two groups were compared,that is,the proportion of patients whose WOMAC score at the end of the 8-week treatment decreased by more than 50%compared to the number of patients before the treatment,as well as the WOMAC osteoarthritis index(0-98),NRS score(0-10),SF-12 quality of life summary(0-100),the proportion of patients taking temporary painkillers,and the safety evaluation.2.Based on the population of 60 patients in the first part,stool samples were collected on an empty stomach once before treatment(week 0)and once after treatment(week 8),and 30 matched healthy subjects were set as the control group to complete defecation.Illumina Miseq high-throughput sequencing technology of 16S rRNA was used to compare the differences of intestinal flora quantity and aggregation between KOA patients and healthy subjects at different levels,and to screen the major pathogenic bacteria groups causing KOA.The changes of intestinal microflora quantity and microflora structure between the electroacupuncture group and the sham acupuncture group after treatment were analyzed,and the differences of the improvement of the electroacupuncture group and the sham group against the main pathogenic bacteria at different levels after treatment were compared.3.Spearman correlation coefficient was used to calculate and analyze the correlation between changes in intestinal flora of patients before and after acupuncture at different levels of phylum,class,order,family and genus and the total WOMAC score,WOMAC pain,NRS score and all clinical indicators.Results1.All the 60 KOA patients completed 8-week acupuncture therapy,and 56(93.3%)completed all clinical trials from baseline 0 weeks to 26 weeks of follow-up.At the end of 8 weeks of acupuncture,the effective response rates of patients in the electro acupuncture group(n=30)and the sham acupuncture group(n=30)were 60%and 30%,respectively,with statistically significant differences(P=0.037).At the 8th week,the NRS score in the electroacupuncture group was significantly lower than that in the sham group(P=0.002),the total WOMAC score and WOMAC pain score in the electroacupuncture group were lower than that in sham group(all P<0.05),the quality of life score SF-12 was increased,no patients in the two groups took temporary painkillers and the incidence of adverse events were low.2.All 90 subjects completed fecal sample collection,including two fecal sample collection for 60 KOA patients before and after treatment and one fecal sample collection for 30 healthy controls.At the level of phylum,compared with healthy subjects,KOA patients showed a decrease in Firmicutes(P=0.034)and Bacteroidetes(P=0.010),and an increase in Proteobacteria(P=0.037).At the class level,the number of Bacteroidia in KOA patients decreased(P=0.010).At the order level,the number of Bacteroidales in KOA patients decreased(P=0.010)and the number of bacterial colonies in lactobacillus was increased(P=0.038).At the family level,the number of Bacteroidaceae decreased in KOA patients(P=0.039),while the number of bacterial colonies in Streptococcaceae increased(P=0.023).At the genus level,the number of patients in KOA decreased in Bifidobacterium,Faecalibacterium,Subdoligranulum,Prevotella,Bacteroides,Blautia,Lactobacillus,Ruminococcus,Agathobacter and Megamonas(all P<0.05).The concentration of Shigella,Streptococcus and Eubacterium_hallii group was high(all P<0.05).Comparison within the group:compared with before treatment,Firmicutes increased and Proteobacteria decreased after electroacupuncture treatment(all P<0.05).Three classes showed significant differences before and after electroacupuncture treatment,namely Clostridia(P<0.01),Negativicutes and Gammaproteobacteria(all P<0.05).Three orders were significantly different before and after electroacupuncture treatment,namely Clostridiales(P<0.01),Enterobacteriales(P<0.01),Selenomonadales(P<0.05).There were significant differences between the five families before and after electroacupuncture treatment,which were Lachnospiraceae,Ruminococcaceae,Entero bacteriaceae,Veillonellaceae and Streptococcaceae(all P<0.05).There were significant differences in five genera before and after electroacupuncture treatment:Faecalibacterium,Ruminococcus and Prevotella were all increased(all P<0.05);Agathbacterium was significantly increased(P<0.01);Streptococcus was decreased(P<0.05).There was no difference in the level of phylum,class,order,family and genus before and after treatment in the sham acupuncture group(all were P>0.05).Comparison between groups:At the genus level,the electroacupuncture group increased Ruminococcus and decreased Escherichia-shigella and Streptococcus compared with sham acupuncture(all P<0.05).At the family level,electroacupuncture increased the number of Ruminococcaceae(P<0.05).3.The correlation between Ruminococcus and WOMAC was negative(P<0.05).The total score of Streptococcus and WOMAC was positively correlated(P<0.05).There was a positive correlation between Escherichia-Shigella and NRS score(P<0.05).The total score of Faecalibacterium and WOMAC pain were negatively correlated(all P<0.01),and negatively correlated with NRS score(P<0.05).The total scores of Agathobacter and WOMAC pain were significantly negatively correlated(all P<0.001),and were significantly negatively correlated with NRS score(P<0.01).The correlation between Escherichia-Shigella and SF-12 was significantly negative(P<0.001).Faecalibacterium was negatively correlated with WOMAC stiffness(P<0.01),negatively correlated with WOMAC function(P<0.05),and positively correlated with SF-12 psychology(P<0.05).Agathobacter showed significant negative correlation with WOMAC function and stiffness(both P<0.01).Conclusion1.Compared with sham acupuncture,8 weeks of electroacupuncture treatment could better reduce the pain degree of patients with knee osteoarthritis.2.Pathogenic bacteria such as Streptococcus and Escherichia-shigella may be potential therapeutic targets and diagnostic biomarkers for KOA.Compared with sham acupuncture,the 8-week electroacupuncture treatment could better regulate the number of beneficial bacteria Ruminococcus in the intestinal tract of KOA patients,and reduce the number of pathogenic bacteria Escherichia-shigella and Streptococcus.3.There was a correlation between the number of intestinal bacteria and the change of bacterial population aggregation and the clinical efficacy:the total scores of beneficial bacteria Ruminococcus and WOMAC were negatively correlated.There was a positive correlation between the total score of WOMAC and Streptococcus. 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