The Change Of Chondrocyte Biological Clock In Osteoarthritis And Its Relationship With Kidney Deficiency Syndrome

Osteoarthritis(OA)is a chronic inflammatory disease with primary cartilage degenerative lesions,and its incidence rate is increasing year by year.Of OA related research shows that in recent years,the occurrence of OA development along with the change of the cartilage cell biological clock,cartilage cell biological clock has the adjustment cartilage wear when "sports"and "resting cartilage repair"of the balance between function,adjust the ability is influenced by the circadian rhythm,when appear circadian rhythm disorders,provides a basis for the OA disease.This theory provides a new idea for the treatment of OA,that is,to regulate the biological clock of chondrocytes to treat OA.Traditional Chinese medicine believes that OA belongs to the category of "bone arthralgia",and kidney deficiency is the key pathogenesis of this disease.In clinical practice,traditional Chinese medicine for tonifying the kidney is used to treat OA with more beneficial effects.Therefore,we wondered whether kidney deficiency was related to the disturbance of chondrocyte clock.Based on this,the purpose of this study was to explore the changes of chondrocyte clock in OA rats and the relationship between the changes of chondrocyte clock and kidney deficiency syndrome in OA rats.Part 1 Changes of chondrocyte biological clock in osteoarthritis ratsObjective To explore the biological clock changes of chondrocytes in osteoarthritis rats.Method 36 6-week old SD rats were randomly divided into the normal group(no intervention),model group(hulth intervention),castration group(hulth+castration intervention)and adenine group(hulth+adenine gavage intervention),9 rats in each group.After successful modeling,the syndrome indexes of rats in each group were detected,and then the samples were taken for the detection of kidney deficiency indexes,including organ index evaluation,E2,T,E2/T,CORT and other pathological indexes related to kidney deficiency,so as to judge the kidney deficiency of rats in each group.The pathological changes of rat cartilage were detected by HE staining and Mankin’s score,to determine whether it met the OA criteria.q-PCR and Western Blot were used to detect the expressions of clock related genes and proteins in chondrocytes,including PI3K,AKT,BMAL1,PER1and CRY1 genes and proteins,so as to observe the changes of chondrocytes’ clock.Result HE staining results showed that the cartilage surface of normal group was smooth,uniform thickness,tidal line intact;the cartilage surface of model group was uneven,thickness became thinner,tidal line disappeared;sham operation group rats showed the same performance as normal group,OA model was successfully prepared;q-PCR results showed that compared with normal group,the expression of bmall gene in model group decreased.The expression levels of perl and pi3k genes increased(P<0.05),akt and cryl genes increased(P>0.05)in the model group,but not in the sham operation group(P>0.05);Western Blot results showed that BMAL1 protein expression decreased,PER1,CRY1 and PI3K protein expression increased(P<0.05)in the model group compared with the normal group.There was an increasing trend in AKT protein in model group(P>0.05),but there was no significant change in sham operation group(P>0.05).conclusion Circadian clock disorder of chondrocytes in osteoarthritis rats.Part 2 To explore the relationship between kidney deficiency and chondrocyte biological clock changes and kidney deficiency syndrome in OA ratsobjective To explore the relationship between kidney deficiency and chondrocyte clock in osteoarthritis rats.Method Thirty-six SD rats aged 6 weeks were randomly divided into normal group(non-intervention),model group(intervention by hulth method),glucosamine hydrochloride group(intervention by hulth method and glucosamine hydrochloride),total flavonoids of Rhizoma Drynariae(intervention by hulth method and total flavonoids of Rhizoma Drynariae),9 rats in each group.After successful modeling,the pathological changes of cartilage were detected by HE staining and Mankin’s score,and the expression of circadian clock-related genes and proteins,including PI3K,AKT,BMAL1,PER1,CRY1 genes and proteins,were detected by q-PCR and Western Blot.Result In the evaluation indexes of kidney deficiency,syndrome score showed that:compared with the normal group,rats in both the adenine group and the castration group presented kidney deficiency syndrome,among which the adenine group presented higher syndrome score(P<0.05),and no kidney deficiency was observed in the model group(P>0.05).Viscera indexes were visible:compared with the normal group,adrenal index and testicular index were increased in the adenine group,with statistically significant differences(P<0.05).Adrenal index increased in the castrated group,but the difference was not statistically significant(P>0.05).No significant increase was observed in the model group(P>0.05).Serological indicators showed that,compared with the normal group,E2 expression was increased,T expression was decreased,E2/T expression was increased,and CORT expression was increased in the castrated group and adenine group,with statistically significant differences(P<0.05).There was no statistical change in the model group(P>0.05).As can be seen from the evaluation of OA indicators,compared with the normal group,HE staining and Mankin’s score in the model group,castrated group and adenine group were all in line with OA performance(P<0.05).As can be seen from q-pcr,compared with the normal group,bmall gene expression decreased in the model group,castration group and adenine group(P<0.05),perl gene expression increased in the castration group,and perl gene and cryl gene expression increased in the adenine group(P<0.05).Compared with the model group,the expression levels of perl gene and cryl gene were increased in the adenine group(P<0.05).Compared with the castrated group,bmall gene expression was decreased in the adenine group(P<0.05).Western Blot analysis showed that,compared with the normal group,the model group,the castrated group and the adenine group showed decreased BMAL1 protein expression(P<0.05),increased PER1 protein expression in the castrated group,increased PER1 protein and CRY1 protein expression in the adenine group(P<0.05),and increased PI3K and AKT gene expression in the adenine group(P<0.05).Compared with the model group,the expression levels of PERI protein and CRY1protein were increased in the adenine group(P<0.05).(P<0.05).Compared with the castration group,CRY protein expression was increased(P<0.05).Conclusion The disturbance of chondrocyte clock in OA rats with kidney deficiency was more serious than that in OA rats,suggesting that kidney deficiency was a factor aggravating the disturbance of OA chondrocyte clock.