| ObjectiveTo explore the therapeutic effect of 1,25-(OH)2D3on osteoarthritis rats and its influence on the structure of intestinal flora,and to explore the effects of changes in the structure and distribution of these flora on osteoarthritis rats Protective effects and related mechanisms that may be involved.Methods:Thirty healthy male SD rats of the same age were selected and randomly divided into 3 groups(n=10),namely model group(OA group),1,25-(OH)2D3 low-dose group(SL group)and High-dose group(SH group).All three groups underwent anesthesia to remove the medial meniscus of the right knee joint to establish an OA model.Gavage was started 2 days after the operation(d),and the low(SL group)and high-dose group(SH group)were given respectively to dissolve in corn Oiled 1,25-(OH)2D3 0.1ug,0.2ug/(kg.d),for 8 weeks,the OA group was given the same amount of corn oil.After 8 weeks of intragastric intervention,venous blood samples of rats were collected through the orbital venous plexus to detect the content of 25-hydroxyvitamin D in the serum;each group of rats was sacrificed,and knee joint specimens of the operated limbs were taken,fixed and decalcified,and frozen sections were made,Perform HE,Safranin and Fast Green staining observation,MMP-13 immunohistochemical analysis,and Mankin score;same Yu Yu gastric administration intervention 8 weeks after the collection of rats feces samples of each group,16S rDNA sequencing analysis,comparison of rats in each group The difference in the composition of the intestinal flora,the screening of the key difference flora,the analysis of the correlation between the key difference flora and the serum 25-hydroxyvitamin D level,and the metagenome function prediction of the intestinal flora of each group of rats with PICRUSt.Result1.Compared with the model group,after 1,25-(OH)2D3 intragastric intervention,the serum 25-hydroxyvitamin D levels of rats in the low-dose group and the high-dose group were significantly increased(P<0.05);Compared with the dose group,the serum 25-hydroxyvitamin D in the high-dose group increased significantly,and the difference between the two was statistically significant(P<0.05).2.HE staining results:large area defects of articular cartilage in the model group,reduced cell numbers,unclear levels,severe proliferation of connective tissue,and erosion of cancellous bone,a large number of inflammatory cells can be seen in the synovial tissue;low-dose group and high-dose group The articular cartilage was locally defective,the thickness increased significantly,the number of cells increased,the hyperplasia of connective tissue was reduced,and some inflammatory cells were seen in the synovium;the results of Safranine Fast Green staining:Large areas of cartilage loss in the knee joints of rats in the model group,and the staining was significantly lighter,There are a large number of inflammatory cells in the synovium,cracks can be seen,and the tide line is unclear.In the low-dose group and the high-dose group,the cartilage layer thickness increased,the loss decreased,the coloration became darker,and the inflammatory cells decreased.3.Compared with the model group,the Mankin score results of the low-dose group and the high-dose group were significantly reduced(P<0.05).Compared with the low-dose group,the score of the high-dose group decreased more significantly,and the difference between the two was statistically significant.Significance(P<0.05).4.Compared with the model group,after 1,25-(OH)2D3intragastric intervention,the ratio of MMP-13 positive cells in the knee joint cartilage of the low-dose group and the high-dose group decreased significantly(P<0.05),and Compared with the low-dose group,the rate of MMP-13 positive cells in the articular cartilage of the high-dose group decreased more significantly,and the difference between the two was statistically significant(P<0.05).5.Compared with the model group,the species diversity in the low dose group had no significant difference(P>0.05),but the species diversity in the high dose group increased significantly(P<0.05).There were significant differences in the structure of intestinal microflora among the three groups.The key differences related to the intervention of 1,25-(OH)2D3 were Bacteroidetes and its subclass horizontal species Bacteroidia,Proteobacteria and its subordinate horizontal species Gammaproteobacteria.Firmicutes and its subclass,order,family,genus level species:Bacilli,Lactobacillales,Lactobacillaceae,Lactobacillus and,Lachnospiraceae NK4A136group.The results of Searpman correlation analysis showed that among the key difference flora related to the intervention of exogenous 1,25-(OH)2D3,the relative abundance of Bacteroidetes and its subordinate class level species of Bacteroidia were significantly negatively correlated with the level of serum 25-hydroxyvitamin D in rats(r<0,P<0.01).he relative abundance of Proteobacteria and its subordinate class level species of Gammaproteobacteria were significantly negatively correlated with the level of serum 25-hydroxyvitamin D in rats(r<0,P<0.01)and There was a significant positive correlation between the relative abundance of Lactobacillus and Lachnospiraceae NK4A136group and the level of serum 25-hydroxyvitamin D in rats(r>0,P<0.01).6.PICRUST results show that there are various potential pathways between intestinal flora and osteoarthritis,and the functional expression of some pathways will be affected after 1,25-(OH)2D3 gavage intervention.Conclusion:1.1,25-(OH)2D3can protect OA by down-regulating the expression of MMP-13 in chondrocytes.2.1,25-(OH)2D3 has a protective effect on osteoarthritis rats by affecting the structure of intestinal microbes.The main mechanism of action may be to inhibit the accumulation of pathogenic bacteria in the intestinal Proteobacteria and Gammaproteobacteria,and promote the growth and colonization of beneficial bacteria such as Lactobacillus and Lachnospiraceae NK4A136group.And restore intestinal health,increase mucosal barrier function,reduce the leakage of inflammatory metabolites and inhibit the production of pro-inflammatory factors,thereby reducing systemic low-grade inflammation,thereby producing a protective effect on osteoarthritis rats. |
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