Apoptosis Of Intervertebral Disc Chondrocytes Through Activation Simvastatin Inhibits Mechanical Stress-induced Of PI3K/AKT Signaling Pathway

Objective:To investigate whether simvastatin(SIM)can inhibit mechanical stress-induced chondrocyte apoptosis by activating the PI3K/AKT signaling pathway,thereby slowing the progression of cervical disc degeneration.Methods:1.6-week SD rats were selected and randomly divided into 4 groups.(1)Bipedal group(Bipedal),(2)Cervical flexion group(Cervical flexion),(3)Low-dose simvastatin group(SIM 20mg/kg),(4)High-dose simvastatin(SIM 40mg/kg).Rats were anesthetized by intraperitoneal injection of 3% pentobarbital(30 mg/kg),and under induced general anesthesia,four groups of rats were constructed as bipedal rats by amputating their tails and forelimbs,and a rat model of anterior cervical flexion compression was constructed using a bending splint.Two of the groups were administered simvastatin simultaneously and the rats were sacrificed after 8 weeks,and the histological structure of the cervical spine was examined by HE staining and immunohistochemistry.2.Human chondrocytes C28/I2 were cultured in vitro,and the cell proliferation viability was detected by CCK8 method at different concentrations of SIM action to determine the concentration of SIM administration in the experiment.3.Human C28/I2 chondrocytes were cultured in vitro,and the protein expression of caspase3,cleaved-caspase3,Bax and Bcl-2 in chondrocytes was detected by Western-blot;apoptosis was detected in each group by flow cytometry.4.Human C28/I2 chondrocytes were cultured in vitro,and the protein expression of PI3 K,P-PI3 K,AKT,and P-AKT in chondrocytes was detected by Western-blot.5.Human C28/I2 chondrocytes were cultured in vitro and given PI3 K inhibitor intervention,and the protein expression of PI3 K,P-PI3 K,AKT,P-AKT,Caspase3,cleaved-caspase3,Bax,and Bcl-2 in chondrocytes was detected by Western-blot;apoptosis was detected by flow cytometry.Results:1.SIM inhibits excessive mechanical stress-induced intervertebral disc degeneration in ratsX-ray test data in the sagittal position of the cervical spine showed that excessive mechanical loading induced a loss of cervical physiological curvature.Chondrocytes in the cervical flexion group were disorganized and reduced in number compared to the bipedal group as detected by HE staining.Gavage treatment with SIM(10 mg/kg and 40 mg/kg,respectively)restored cervical curvature,improved histological damage,and ameliorated degeneration of CEP in rats induced by mechanical loading,indicating the potential value of SIM in the treatment of IDD.2.Measurement of SIM administration concentration in C28/I2chondrocytesTo estimate the cytotoxicity of SIM and its effect on chondrocyte apoptosis,a CCK-8 cell viability assay was performed.The CCK-8 results showed that no significant cytotoxicity was observed in the SIM treatment group at concentrations less than 40 μM after 24 h of co-culture with chondrocytes.In contrast,cell viability gradually decreased in SIM exposed to higher concentrations(40-160 μM).3.SIM inhibits mechanical stress-induced chondrocyte apoptosisTo investigate the effect of SIM on mechanical loading-induced apoptosis in chondrocytes,Western blotting was performed in vitro to assess the expression of apoptosis-related proteins in chondrocytes.It was found that under abnormal mechanical loading in vitro,SIM decreased the expression of apoptosis-related proteins,such as Bax,caspase3 and cleaved caspase3,and increased the expression of anti-apoptotic proteins,such as Bcl-2.Flow cytometry results showed that SIM decreased the apoptosis rate of chondrocytes under mechanical loading of 0.5 MPa.Therefore,the protective activity of SIM against mechanical load-induced CEP degeneration may be related to the inhibition of chondrocyte apoptosis.4.SIM activates PI3K/AKT signaling pathway in chondrocytes stimulated by mechanical stressIn vivo studies revealed that PI3 K and AKT expression was up-regulated by immunohistochemical detection by SIM.Consistently,the expression of PI3 K and AKT at the m RNA and protein levels was also significantly upregulated by SIM in mechanically loaded treated C28/I2 cells.In addition,SIM enhanced the expression of Bcl-2 and inhibited the expression of Bax,caspase3 and cleaved caspase3 in vitro.In conclusion,the ameliorating effect of SIM on mechanical loading-induced chondrocyte apoptosis may be related to the upregulation of PI3K/AKT expression.5.SIM inhibits mechanical stress-induced chondrocyte apoptosis through activation of PI3K/AKT signaling pathwayIn mechanically loaded treated chondrocytes,LY294002 inhibited the activity of the SIM-activated PI3K/AKT signaling pathway,as evidenced by reduced phosphorylation of PI3 K and AKT.In addition,LY294002 also attenuated the ameliorating effect of SIM on chondrocyte apoptosis,which could be confirmed by partial restoration of apoptosis-related protein expression and apoptosis rate detected by flow cytometry.Thus,SIM activates PI3K/AKT signaling and inhibits chondrocyte apoptosis induced by mechanical loading.Conclusion:SIM inhibits mechanical stress-induced apoptosis of disc chondrocytes and delays disc degeneration by activating the PI3K/AKT signaling pathway.