| Objective:The purpose of this study was to observe the clinical efficacy of shaoyang xibi decoction in the treatment of knee osteoarthritis with deficiency of liver and kidney,and based on the miR-15a-5p/Akt pathway to preliminary explore the mechanism of Shaoyang Xibi decoction in treating osteoarthritis,so as to provide a basis for the clinical application of the theory of"shaoyang dominating bone".Method:(1)Seventy-two patients with knee osteoarthritis with deficiency of liver and kidney were randomly assigned to the experimental group,celecoxib group and xianling gubao group,with 24 patients in each group.Patients in each group were evaluated with WOMAC score,quantitative standard score of TCM syndrome of osteoarthritis and score of shaoyang syndrome after the corresponding treatment,and the changes of each score were observed and the efficacy of TCM was evaluated.(2)miRBase database and TargetScan gene bank were screened for related miRNAs as observation indicators.24 SD rats were randomly divided into groups A,B and C according to the random number table method.Two groups B and C were modeled by Hulth method.Group A:blank group,rats were given normal saline by gavage.Group B:in the experimental group,rats were given shaoyang xibi decoction by gavage after modeling.Group C:control group,rats were given normal saline by gavage after modeling.After 4 weeks,the cartilage of the knee joint of the rats was stained with HE to observe the pathological changes.The contents of IL-1?and TNF-? in articular fluid were determined by Elisa.RT-PCR was used to detect the relative expressions of miR-15a-5p,miR-145-5p and miR-29b-3p in the cartilage of the knee joint of rats.(3)Three groups of cartilage with normal or different degrees of degeneration were divided into normal group,mild group and severe group according to the degree of injury.RT-PCR was used to detect the expression of miR-15a-5p and Akt-mRNA in each group.The apoptosis rate of chondrocytes in each group was determined by Tunel method.The correlation between miR-15a-5p and the expression level of Akt-mRNA and the apoptosis rate of chondrocytes was analyzed.(4)The pmirGLO plasmid was treated with Sac I and Sal I double enzyme digestion,and the 3'-UTR of the mRNA containing wild type(WT)and mutated type(MUT)Akt was cloned onto the plasmid after enzyme digestion to construct the double luciferase vector pGL3.The two constructed pGL3 vectors were transfected with either a miR-15a-5p-mimic or a miR-15a-5p-NC,respectively,and the targeting of miR-15a-5p in the regulation of Akt-mRNA was verified by luciferase reporter assay.Then,miR-15a-5p-mimics,miR-15a-5p-NC and miR-15a-5p-inhibitors were transferred into SW1353 cells,respectively,and the expressions of Akt,p-Akt,IkB-a,p-IkB-a,p65,p-p65 and mTOR,p-mTOR were detected by Western Blot.Normal SW1353 cells and SW1353 cells treated with miR-15a-5p-mimic or miR-15a-5p-NC were collected,and the apoptosis rate and cell cycle were detected by flow cytometry.Results:(1)The score of WOMAC in the experimental group after treatment with shaoyang xibi decoction was lower than that before the treatment,and there was no significant difference between the xianling gubao group and the celecoxib group.The score difference of WOMAC before and after treatment in the experimental group was lower than that of celecoxib group and higher than that of xianling gubao group.In the experimental group,the standard score of TCM syndrome quantization after treatment with shaoyang xibi decoction was significantly reduced,which was better than that of celecoxib group,and there was no significant difference between the xianling gubao group.The difference of TCM syndrome score between the experimental group and the xianling gubao group was higher than that between the celecoxib group,and there was no difference between the experimental group and the xianling gubao group.Among the 72 patients observed in this study,67(93.06%)had shaoyang syndrome,shaoyang xibi decoction could significantly reduce the score of shaoyang syndrome after treatment,which was better than the celecoxib group and xianling gubao group.The effective rate of the experimental group was 95.65%.The effective rate of celecoxib group was 78.26%.The effective rate of xianling gubao group was 91.67%.The experimental group was superior to the celecoxib group and there was no significant difference between the two groups.(2)The pathological results confirmed that the cartilage degeneration of the knee joint of the rats after Hulth model was different from that of the blank group,and the cartilage degeneration of the rats in the experimental group was less severe than that in the control group.The levels of IL-1? and TNF-? in the articular fluid of rats in the experimental group were lower than those in the control group and higher than those in the blank group.The expressions of miR-15a-5p,miR-29b-3p and miR-145a-5p in the cartilage of rats in the experimental group and control group were significantly different from those in the blank group,among which miR-15a-5p and miR-29b-3p were up-regulated and miR-145a-5p was down-regulated.The expression of miR-15a-5p in the experimental group was lower than that in the control group.However,miR-29b-3p and miR-145a-5p were not differentially expressed in the experimental group and the control group.(3)The detection results of RT-PCT showed that the relative expression of miR-15a-5p in osteoarthritis cartilage was increased,and the expression level of the severe group was higher than that of the mild group.The relative expression of Akt-mRNA in osteoarthritis chondrocytes decreased,and the expression of Akt-mRNAin the severe group was lower than that in the mild group.The apoptosis rate of osteoarthritis chondrocytes in the severe group was higher than that in the mild group,and the lowest in the control group.Correlation analysis showed that the relative expression of miR-15a-5p in chondrocytes was negatively correlated with the expression of Akt mRNA.The relative expression of miR-15a-5p in chondrocytes was positively correlated with the apoptosis rate.(4)A luciferase gene report confirms that miR-15a-5p can target Akt mRNA.Western Blot results confirmed that over-expressed miR-15a-5p can inhibit the expression of Akt pathway-related proteins in the cell,while the inhibition of miR-15a-5p expression by Shaoyang Xibi decoction can increase the expression of Akt-related proteins in the cell.The results of flow cytometry showed that the apoptosis rate of cells transferred to miR-15a-5p-mimics increased by 6.22 times compared with that of normal SW1353 cells,and most of the cells remained before G1 phase.The high expression of miR-15a-5p blocked the cells in the G0/G1 phase and induced apoptosis.Conclusions:(1)Shaoyang xibi decoction can effectively treat knee osteoarthritis with deficiency of liver and kidney,especially for patients with deficiency of liver and kidney combined with shaoyang syndrome.(2)Shaoyang xibi decoction can delay cartilage degeneration in osteoarthritis rats,reduce cartilage inflammation and inhibit the expression of miR-15a-5p in chondrocytes.(3)miR-15a-5p was highly expressed in osteoarthritis cartilage,negatively correlated with Akt mRNA,and positively correlated with apoptosis rate.(4)After inhibiting the expression of mir-15a-5p,shaoyang xibi decoction can increase the expression of Akt mRNA and reduce the apoptosis of chondrocytes through the Akt pathway. |
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