| Objective Through animal experiments and cell experiments,the role of electroacupuncture at xiyan Points in inhibiting cartilage degeneration of knee osteoarthritis was investigated from Rho A/ROCK signaling pathway,and the possible targets of electroacupuncture to delay articular cartilage degeneration were further explored.Methods 1.Animal experiment: Rat model of knee osteoarthritis was established by modified Hulth operation and randomly divided into 5 groups,namely normal group,model group,electroacupuncture knee group,electroacupuncture non-acupoint group and inhibitor group.Rats in the normal group were not given any intervention.The model group and the inhibitor group were given the same grasping and sock fixation for 15 minutes during the electroacupuncture of the two electroacupuncture groups.The electroacupuncture knee group was given the internal and external knee electroacupuncture for 15 minutes.The needles were given acupuncture at a non-acupoint group for 15 minutes as a control.Rats in the inhibitor group were injected with 0.1 ml of Y-27632 solution once a day,once a day for 5 days,and rested for 2 days.Injection for 12 weeks.The two groups of electroacupunctures were set to the sparse wave mode,the pulse output frequency was 2/100 Hz,and the intensity was 0.5-1.5 m A.The local muscle began to twitch and the rats did not struggle.Intervention 1 time,intervention for 5 days per week,a total of 12 weeks of intervention.After the intervention,the right knee joint cartilage tissue was collected.HE staining and saffron green staining were used to observe the morphological changes of cartilage.The apoptosis of chondrocytes was detected by TUNEL method.Cartilage was detected by RT-q PCR and Western Blot.Expression of related m RNA and protein of Rho A,Rock,ERK1/2,Bax and Bcl-2 in Rho A/Rock signaling pathway.2.Cell experiment: Establish chondrocyte culture system in vitro,type II collagen and toluidine blue staining method to identify chondrocytes,MTT method to detect the growth of the first F1?F3 chondrocytes,and draw a growth curve,preferably chondrocytes.After well-cultured chondrocytes were cultured 72,they were randomly divided into blank group,model group,electroacupuncture knee group,electroacupuncture non-acupoint group and inhibitor group.Normal group: 100 ul of medium containing 10% normal group serum;model group: TNF-?20ng/m L+10% model group serum medium total 100ul;electroacupuncture knee group: TNF-?20ng/m L+10% Electroacupuncture and knee group blood serum a total of 100ul;electroacupuncture non-acupoint group: TNF-?20ng/m L+10% electroacupuncture non-acupoint group serum medium a total of 100ul;inhibitor group: added TNF-?20ng / m L + inhibitor Y-27632 10 u M + 10% model group serum medium a total of 100 ul.After 4 hours of intervention,the activity of chondrocytes in each group was detected by CCK-8 method.The apoptosis of chondrocytes was detected by Annexin V flow cytometry.The Rho A/Rock signal transduction pathway of chondrocytes was observed by RT-q PCR and Western blot.Expression of related m RNA and protein in Rho A,Rock,ERK1/2,Bax and Bcl-2.Results 1.Animal experiment: HE staining and safranin solid staining of articular cartilage tissue in rats with knee osteoarthritis model were significantly improved compared with model group or electroacupuncture non-acupoint group.There was no significant difference between the two groups in the acupuncture and knee group and the inhibitor group.The TUNEL test showed that the electroacupuncture group and the inhibitor group could significantly reduce the chondrocyte apoptosis index compared with the model group or the electroacupuncture group.(P<0.05),the difference between the electroacupuncture group and the inhibitor group was not statistically significant(P>0.05);RT-q RCP and Western Blot results showed that compared with the model group or electroacupuncture non-acupoint group,electricity The expression levels of key genes and proteins such as Rho A,Rock,ERK1/2 and bax were down-regulated in the acupuncture and knee group and the inhibitory group,and the expression of Bcl-2 gene and protein was up-regulated(P<0.01).There was no statistically significant difference between the electroacupuncture group and the inhibitor group(P>0.05).2.Cell experiment: Chondrocytes stained with type II collagen showed cytoplasmic staining as brown-yellow particles,which were in the form of flakes or clumps;stained with toluidine blue,and purple-red heterochromic particles in the cytoplasm.According to the characteristics of the growth curve of the F1 to F3 chondrocytes,it is preferred to perform cell experiments within 3 to 6 days after the F2 chondrocytes are plated.Intervention at 20 ng/ml for 4 h was the best dose and time effect of TNF-?-induced chondrocyte apoptosis.After intervention in each group,the chondrocyte viability of the electroacupuncture group and the inhibitor group was significantly higher than that of the model group or the electroacupuncture group(P<0.05),but the electroacupuncture group had no difference compared with the inhibitor group.Statistical significance(P>0.05).The results of flow cytometry showed that the total apoptotic rate of electroacupuncture group and inhibitor group was significantly lower than that of model group or electroacupuncture group(P<0.05),total electroacupuncture group and model group.There was no statistical difference in apoptotic rate(P>0.05).It indicated that TNF-? could significantly promote chondrocyte apoptosis after intervention of chondrocytes(P<0.01),while sera after electroacupuncture could inhibit TNF-?-induced chondrocyte apoptosis.The results of RT-q RCP and Westernblot showed that the expression levels of Rho A,Rock,ERK1/2 and Bax in the electroacupuncture group and the inhibitor group were significantly lower than those in the model group or electroacupuncture group(P<0.05);Bcl-2 gene,protein expression and Bcl-2/Bax ratio in chondrocytes of electroacupuncture group were significantly higher than those in model group or electroacupuncture group.There were no significant differences in Rho A,Rock,ERK1/2,Bax,Bcl-2 gene and protein expression between the electroacupuncture group and the inhibitor group,and between the electroacupuncture group and the model group(P >0.05).Conclusion 1.Electroacupuncture at xiyan Point can effectively alleviate the damage of knee osteoarthritis on rat cartilage tissue morphology.Electroacupuncture at knee acupuncture points regulates Rho A/Rock signal transduction pathway and down-regulates Rho A,Rock,ERK1/2,bax The expression levels of key genes and proteins,up-regulation of Bcl-2 gene and protein expression levels,inhibition of articular chondrocyte apoptosis,reduce cartilage destruction,maintain the relative integrity of cartilage structure,thereby delaying the degeneration of articular cartilage.2.The electroacupuncture of the xiyan Point inhibits the transmission of Rho A/Rock signaling pathway in chondrocytes,down-regulates the expression levels of Rho A,Rock,ERK1/2,Bax and other genes and proteins in the Rho A/Rock pathway of chondrocytes,and up-regulates the Bcl-2 gene.And the expression level of the protein,thereby inhibiting the apoptosis of chondrocytes and delaying the degeneration of chondrocytes. |
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