| Objective: To observe the clinical efficacy of the ligament-skeleton sequential manipulation in the treatment of knee osteoarthritis,and to detect the changes of local tibial bone mineral density(BMD)and 3 representative serum markers of bone metabolism including bone glaprotein(BGP),procollagen type I N-terminal propetide(P1NP)and tartrate-resistant acid phosphatase(TRACP)before and 6 months after treatment,and to investigate the mechanism of the manipulation on bone metabolism in combination with the intestinal microecology.Methods: A total of 60 patients with primary knee osteoarthritis were selected and randomly divided into the ligament-skeleton sequential manipulation treatment group and the conventional manipulation control group,30 cases in each group.Both groups were observed in the changes of WOMAC scores and the Lysholm knee scores before,after and6 months after treatment and the differences were compared.DXA was used to measure the region of interest(ROI)on the lateral tibia,both groups were compared in BMD of tibial subchondral before and 6 months after treatment,and the differences were compared.Enzyme-linked immunosorbent assay(ELISA)was used to detect the changes of BGP,P1 NP and TRACP before and 6 months after treatment in the 2 groups,and the differences between were compared.16 sr RNA gene sequence was used to sequence the gut microbiota in the feces of both groups before treatment and the blank healthy control group,and the quality analysis was conducted to compare the differences between the sample groups.LEf Se was used to screen the key species of the community,and the different species were compared with KEGG database to predict the function of the different gut microbiota.The screened differential bacteria were infused into the stomach of mice induced by MIA,and the mice in the treatment group were subjected to knee flexion and extention.Bone sections and CT scans of the knee joints were observed after execution.Results:(1)Both the treatment group and the control group after the treatment had reduced WOMAC scores,enhanced Lysholm knee scores than those before treatment,and the difference was statistically significant(P<0.05);Both groups at 6-month follow-up after treatment the WOMAC scores increased,but lower than that before treatment;and reduced Lysholm knee scores compared with those at the end of treatment.(2)Both groups had the tibial BMD compared before and 6 months after treatment,and the score difference within the treatment group was statistically significant(P < 0.05),while the difference within the control group was not significant.Both groups had serum bone metabolism markers BGP,P1 NP and TRACP levels compared before and 6 months after treatment,and the treatment group had both increased BGP and P1 NP,while decreased TRACP,with statistically significant differences in scores within the group(P < 0.05),but no significant differences within the control group.A total of 2,017 OTUs were obtained from 94 samples,of which 1,873 were obtained from the KOA group and 1,696 from the HC group,the 2groups had 1552 overlapping OTUs.Fecal samples in both groups had the main gut microbiota of bacteroidetes,firmicutes,actinobacteria and proteobacteria.Both groups of samples had the top 4 in the order of bacteroidetes,prevotella,escherichia and faecalibacterium at the genus level.The KOA group had significant enrichment of faecalibacterium,atopobium,phascolarctobacterium.,and simultaneously the KOA group had significantly reduced megamonas,cyanobacteria and prevotellaceae.Genes related to metabolism were significantly reduced in the KOA group,with the difference statistically significant(P<0.05),mainly including glucose metabolism,lipid metabolism,amino acid metabolism and metabolism.(3)Both of the mice gavaged with Faecalibacterium showed no significant changes before and after treatment on CT,and no obvious structural changes in the tibia of mice with the HE dye.Conclusion:(1)Both the ligament-skeleton sequential manipulation and conventional manipulation are effective in the treatment of knee osteoarthritis.The ligament-skeleton sequential manipulation is better than the conventional manipulation in reduction of WOMAC scores and Lysholm knee scores.The treatment group has better long-term curative effects than the control group in delaying the pain,stiffness and daily activities due to KOA degeneration,indicating that the ligament-skeleton sequential manipulation has a certain long-term curative effect.(2)The ligament-skeleton sequential manipulation can increase the subchondral BMD of KOA patients,but it is not significant in the conventional manipulation group.KOA patients in the treatment group has improved systemic bone metabolism,indicating that the osteogenesis is more than the bonehew(bone resorption).No difference is observed in the abundance of gut microbiota in patients with knee osteoarthritis compared with normal people,but there is significant difference in the composition of the gut microbiota.In this study,it has been found that there are significant differences between gut microbiota in the KOA group and the healthy group,mainly due to the obvious enrichment of faecalibacterium,atopobium,and phascolarctobacterium in the KOA group.However,megamonas,cyanobacteria and prevotellaceae are significantly reduced in the KOA group.Faecalibacterium,phascolarctobacterium and megamonas can be used as biomarkers to distinguish patients with knee osteoarthritis from healthy people.Gut microbiota in patients with knee osteoarthritis may be related to the 3 major metabolic pathways,and the metabolic pathways may affect the occurrence and progression of knee osteoarthritis.(3)The effect of flexion and extention to simulate the ligament-skeleton sequential manipulation in MIA mice is not obvious. |
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