| Objective Based on the BMP2/ Smad8/ Runx2/ Osterix pathway,the time-sequence effect on the differentiation of osteoblasts was studied through the experimental observation of RA rats with Zhuang medicine Longzuan Tongbi decoction and the combination with animal morphology,pathology,protein and gene detection experiments.Methods:96 male SD rats were randomly divided into model group,experimental group,control group and normal group,with 24 rats in each group and Adjuvant Arthritis.The observation time was 28 days,divided into 4 stages,one stage for 7 days.Six rats would be killed at the end of each stage in four groups.The dosage of the experimental group was 0.27g/ 100g/ d,with the control group 0.9mg/kg/d,and the normal group and the model group were given the same dose of distilled water every day.The drug was discontinued for 1 week from the 1st day of the 4th stage,and the last batch was taken at the 28 th day.Western blotting and Real-time PCR were used to detect the expression of BMP2,smad8,Runx2 and osterix in the synovium of the knee joint of RA rats;BMP2,Smad8,Runx2 and Osterix protein in the serum were tested by enzyme-linked immunosorbent assay(ELISA).At last,the ankle joint of rats Pathological changes were observed through HE staining.Results:24 out of 96 SD rats were normal,while the rest of them were successfully modeled without death.The hair color,claw nail,diet,mental state and defecation of the model group were lower than those of the normal group.The most obvious change was found in the experimental group near the 21 d period,and there was no significant difference between the control group.2 Pathological changes of rat knee joint:HE staining results showed that after 21 days later,the synovium,articular cavity and osteoblasts of the normal group were all normal.Compared with the normal group,the model group had synovial hyperplasia,less smooth surface,fewer osteoblasts,disordered order,and a large number of inflammatory exudate associated with lymphocytes in the joint cavity.Compared with the model group,after 21 d,no obvious inflammatory exudate and stenosis were observed in the articular cavity of the experimental group,with clear and smooth synovial layer,orderly arrangement of osteoblasts and columnar distribution.In the control group,there were more osteoblasts,no obvious stenosis in the articular cavity,no obvious hyperplasia and edema in the synovial membrane,and the cartilage layer was repaired to some extent.WB results showed that the expression of BMP2,Smad8,Runx2 and Osterix proteins in the normal group and the model group showed no temporal difference in the whole period.While their expression in the model group was weak at all stages.At 21 days,it was significantly up-regulated(P < 0.05).But Runx2 showed the highest expression in 14 days.The trend of protein difference in each time period of the control group was similar to that of the experimental group.Compared with the normal group,the serum levels of BMP2,Smad8,Runx2 and Osterix in the model group decreased significantly(P < 0.05).In the four time periods,the protein levels of BMP2,Smad8,Runx2 and Osterix in the experimental group and the control group were the lowest in the 7-day period.The expression of BMP2,smad8 and osterix was the strongest at 21d(P < 0.05),and the expression of Runx2 serum protein was weaker at 21 d than 14 d.There were no temporal differences in BMP2 m RNA,Smad8 m RNA,Runx2 m RNA and Osterixm RNA between the normal group and model group,while it was obviously different between the experimental group and control group.The expressions of BMP2 m RNA,Smad8 m RNA and Osterixm RNA in the experimental group and control group were the weakest at 7d,significantly up-regulated at 14 d and strongest at 21d(P < 0.05).Runx2 m RNA was slightly weaker at 21 d than it at 14 d,but still higher than it at 28 d.There was no significant difference in time sequence between the control group and the experimental group.Conclusion:1.Longzuan Tongbi decoction has a bone-protective effect on RA rats,and the mechanism may be related to the activation of the BMP2/Smad8/Runx2/Osterix pathway to promote osteoblast differentiation.2.The effect of Longzuan Tongbi decoction on promoting osteoblasts differentiation may begin to emerge on 7th day,and the peak effect may occur on 21 st day.3.3.The effect of Longzuan Tongbi decoction on the differentiation of osteoblasts in RA rats may be significant from the 7th day,and the peak value of the effect may be around 21 d. |
