The Role Of High Fat Diets And Exercise Regulating Gut Microbiota And LPS-TLR4/NF-?B Signaling In Osteoarthritis In Mice

BackgroundObesity is an important factor inducing osteoarthritis.Recent studies have found that the imbalance of intestinal flora and the activation of metabolic and immune pathways are closely related to the formation of osteoarthritis.In vitro experiments showed that the abnormal expression of lipopolysaccharide?LPS?,and its mediated toll-like receptor 4?TLR4?signaling caused chondrocyte injury.Increased expression of TLR4 in the articular cartilage of patients with osteoarthritis was also found.However,whether dysbacteriosis and the LPS-TLR4/NF-?B signaling pathway is involved in the occurrence of obese osteoarthritis induced by high-fat diet,and whether exercise can regulate the intestinal flora and its downstream molecular signaling pathway in obese patients and affect the pathological process of osteoarthritis have not yet been reported.1^st part:MethodsThe male C57BL/6J mice were randomly divided into normal control group?n=6?and high fat group?n=10?according to their body weight.Weight and fasting blood glucose were monitored weekly.Eight weeks later,the mice in the high-fat diet group whose body weight was higher than 20%of the average weight of mice in the normal diet group were selected as obese mice.The mice in normal control group?NC group,n=6?and high-fat obese group?HF group,n=6?were sacrificed.The left knee joint was fixed,decalcified and sectioned with paraffin,and evaluated by OARSI score and matrix proteoglycan content.Results:Pathological changes were observed in cartilage of keen-joints in mice of HF group.The OARSI score of of medial femoral condyle,lateral femoral condyle,medial tibial plateau and lateral tibial plateau of knee cartilage in the HF group mice was significantly higher than that of the NC group.Both the superficial layer and the transition layer in four positions of the cartilage were stained shallower than that in mice of NC group.Conclusions:The morphological score of knee-joint OARSI in HF group was significantly increased,and the histomorphologic and pathological changes were similar to those in the previous OA model.The model of obesity-associated OA in mice was successfully established after 8-week high-fat diet.2^nd part MethodsThe male C57BL/6J mice were randomly divided into normal control group?n=12?and high fat group?n=20?according to their body weight.Eight weeks later,the mice in the high-fat diet group whose body weight was higher than 20%of the average weight of mice in the normal diet group were selected as obese mice.The obese mice were randomly divided into 2 groups:high-fat diet sedentary group?HS group,n=6?and the high diet with voluntary exercise group?HE group,n=6?.The normal group were randomly divided into normal diet sedentary group?CS group,n=6?,the normal diet with voluntary exercise group?CE group,n=6?.The mice in the exercise group had access to wheel.Weight and fasting blood glucose were monitored weekly,and all experimental animals were sacrificed after 4 weeks.The left knee joint was fixed,decalcified and sectioned with paraffin,and evaluated by OARSI score and matrix proteoglycan content.Bioinformatics analysis of intestinal flora was performed.LPS levels in serum and articular fluid were measured with endotoxin detection kit.The cartilage of the knee joint of the right hind limb of mice was ground into bone powder to extract total protein.The expression of TLR4,MyD88,NF-?B P65 and MMP13protein in cartilage were detected by Western immunoblot.Results:Histomorphology:?1?After 4 weeks of voluntary wheel exercise,body weight and blood glucose in the HE group was significantly lower than that in the HS group.There was no significant difference in the daily exercise distance between the mice in CE group and that in HE group.?2?There was no significant correlation between body weight and OARSI score of articular cartilage in mice.?3?The OARSI scores of the medial femoral condylar,lateral femoral condylar and medial tibial plateau in the HS group mice were significantly higher than that in CS group;these OARSI scores in HE group mice were significantly lower than that in HS group;these OARSI scores in HE group mice were significantly higher than that in CE group.?4?There was no interaction between diet factors and exercise factors on the OARSI score of the lateral tibial plateau cartilage in mice.?5?In CE group mice,the superficial layer,transitional layer and radial layer of the medial and lateral condyles of femur and the medial and lateral cartilages of tibial plateau were stained more deeply.In HS group mice,the superficial layer,transitional layer and radial layer of cartilage were all stained shallowly.Compared with HS group mice,the superficial layer,transitional layer and radiation layer of cartilage in HE group mice were more stained.Bioinformatics analysis of intestinal mirobiota:?1?Shannon diversity index curve and Coverage index curve tended to be flat.?2?The Venn figure shows that different intervention measures make certain differences in the composition and structure of bacterial flora.?3?The NMDS analysis showed the different intervention measures make certain differences in the composition and structure of bacterial flora.?4?PCoA results showed HS group and HE group were far away from CS group and CE group.There was also a large interval between HS group and HE group.?5?The Ace index and Chao1 index of intestinal flora of mice in HS group were significantly lower than those in CS group.The Ace index and Chao1index of intestinal flora in HE group were significantly higher than those in HS group.?6?Dietary and exercise factors had no reciprocal action on intestinal flora evenness Shannon index and Simpson index.?7?The abundance of intestinal Firmicutes in HS group was significantly higher than that in the CS group.Those microbiota in the HE group was significantly lower than that in the HS group.?8?Dietary and exercise factors had no major effect on abundance of Bacteroidetes in the intestinal tract of mice.?9?The ratio of Firmicutes/Bacteroidetes in HS group was significantly higher than that in the CS group.The ratio in the HE group was significantly lower than that in the HS group.?10?The bacterial abundance of Desulfovibrionaceae in intestinal of HS group mice was significantly higher than CS group.Those microtiota in the intestinal tract of mice in the HE group was significantly lower than that in the HS group.?11?The abundance of Bifidobacteria and Bacteroidales_S24-7 in HS group mice is significantly lower than that in CS group mice.Those microtiota in the intestinal tract of CE group mice was higher than that in CS group mice.?12?The LPS level of joint fluid was positive correlated with the OARSI score of medial femoral condyle,lateral femoral condyle,medial tibia platform and lateral tibia platform.LPS-TLR4/NF-?B signalling:?1?The LPS levels of joint fluid in HS group mice were significantly higher than that in CS group mice.The LPS levels in HE group mice were significantly lower than that in HS group mice.?12?The LPS level has a positive correlation with the abundance of Desulfovibrionaceae,and a negative correlation with the abundance of Bifidobacteria and Bacteroidales_S24-7.?2?TLR4protein expression of knee cartilage in HS group mice was significantly higher than that in CS group mice.The expression of TLR4 protein in HE group was significantly lower than that in HS group mice.?3?The expression of MyD88protein in knee cartilage in HS group was significantly higher than that in CS group mice.MyD88 protein expression in HE group was significantly lower than that in HS group mice.?4?Dietary and exercise factors had no reciprocal action on expression of NF-?B P65 protein in knee cartilage in mice.?5?The expression of MMP13 protein in HS group mice was significantly higher than that in CS group mice.The expression of MMP13 protein in knee cartilage in HE group mice was significantly lower than that in HS group mice.Main conclusions:?1?Intestinal disorders induced by a high-fat diet in obese mice may have an important influence in the OA disease.The excessive activation of LPS-TLR4/NF-?B signaling in articular may involve in obesity-associated OA.?2?Although not completely reversed,exercise alleviated the severity of obesity-associated OA in mice may through regulating intestinal flora and reducing the overactivation of LPS-TLR4/NF-?B signaling.