| Objective:Intervertebral disc degeneration is mainly characterized by "lumbago",which belongs to "arthralgia" in Chinese medicine.Kidney deficiency and blood stasis is its main pathogenesis.As a driving factor of oxidative stress,inflammatory factor infiltration can induce imbalance of intracellular energy metabolism and damage of mitochondrial function,and then cause apoptosis of nucleus pulposus cells and degradation of extracellular matrix,eventually leading to intervertebral disc degeneration.Based on the principle of "Tonifying kidney and Huoxue",Bushen Huoxue Formula has a good clinical effect in the treatment of intervertebral disc degeneration,but the specific mechanism of action is not detailed.In vitro experiments were conducted to verify the pathogenesis of intervertebral disc degeneration caused by inflammatory factors through inducing nucleus pulpoile cell apoptosis,and the main purpose of this study was to explore the mechanism of inhibiting intervertebral disc degeneration by Bushen Huoxue Formula based on AMPK/SIRT1 signaling pathway.Method:Nucleus pulposus tissues of clinical patients were collected for isolation,extraction and culture.To observe the protective effect of Bushen Huoxue Formula on nucleus pulposus cells by TNF-α intervention.(1)CCK-8 method was used to detect the effect of BSHXF and TNF-α on the proliferation of nucleus pulposus cells;(2)The gene expression levels of MMP-3,MMP-9,ADAMTS-4,ADAMTS-5 were detected by real-time PCR;(3)The expression of collagen II and MMP-3 was detected by immunofluorescence staining.(4)The protein expression levels of Collagen II,Aggrecan,ADAMTS-4,MMP-3,Cleaved-caspase 3,mito-cyt,cyto-cyt were detected by Western Blot.(5)TUNEL was used to detect the apoptosis of nucleus pulposus cells under the intervention of TNF-α and BSHXF.(6)The morphology of mitochondria and apoptotic bodies were observed by transmission electron microscopy.(7)Flow cytometry was used to detect the apoptosis of nucleus pulposus cells under the intervention of TNF-α and BSHXF;(8)ROS content and mitochondrial localization in nucleus pulposus cells were detected by reactive oxygen species(ROS)and mitochondrial fluorescence probe;(9)SOD activity of nucleus pulposus cells was detected by Cu Zn/Mn-SOD method;(10)ATP metabolism of nucleus pulposus cells was detected by ATP chemiluminescence method.(11)The expression of AMPK in AMPK/SIRT-1 signal pathway was detected by Western blot.Result:1.CCK-8 method was used to detect the proliferation of nucleus pulposus cells under the intervention of BSHXF medicated serum and TNF-α:(1)Compared with the blank serum group,the proliferation rate of nucleus pulposus cells treated with different concentrations of BSHXF medicated serum increased(P <0.01)in 5%、10%、15% and 20% BSHXF medicated serum group,and the best concentration was 15% BSHXF medicated serum(P <0.01);(2)Compared with the blank serum group,the cell viability of 5%、10%、15% and 20% blank serum group increased after intervention with different concentrations of blank serum for 24 h(P <0.05),but there was no significant difference in the proliferation rate among the blank serum treatment groups(P> 0.05),indicating that the concentration change of blank serum did not affect the proliferation rate of nucleus pulposus cells.(3)After 24 hours of TNF-αintervention,the proliferation rate of model group(TNF-α intervention)was lower than that of blank control group(P <0.01).Compared with the model group(TNF-α intervention),the increment rate of 5%、10% and 15% BSHXF medicated serum group were higher(P <0.01).2.The gene expression levels of MMP-3,MMP-9,ADAMTS-4,ADAMTS-5 in model group were significantly higher than those in control group(P <0.01).Compared with the model group(TNF-α intervention),the gene expression levels of ADAMTS-4,ADAMTS-5,MMP-3,MMP-9 in 15% BSHXF group were significantly decreased(P <0.01).3.The expression of Collagen II and MMP-3 in model group was lower than that in control group(P <0.01),and the expression of MMP-3 was higher than that in control group(P<0.01).Compared with the model group(TNF-α intervention),the expression level of Collagen II in 15% BSHXF group was increased(P <0.01),and the expression level of MMP-3 was decreased(P <0.01).4.Western Blot showed that the expression of Collagen II,Aggrecan,mito-cyt,ADAMTS-4,MMP-3,Cleaved-caspase-3,mito-cyt,c-caspase-3 in model group was significantly lower than that in control group(P <0.01),and the expression of ADAMTS-4,MMP-3,c-caspase-3,mito-cyt,c-caspase-3 was significantly higher than that in control group(P<0.01).Compared with the model group(TNF-α intervention),the protein expression levels of Collagen II,Aggrecan and mito-cyt in the 15% BSHXF group were increased(P<0.01).The expression of ADAMTS-4,MMP-3,cyto-cyt and c-caspase-3 protein decreased(P <0.01).5.The apoptosis of nucleus pulposus cells was detected by TUNEL.Compared with the control group,the number of apoptosis cells in model group(TNF-α intervention)was increased(P <0.01).Compared with the model group(TNF-α intervention),the number of apoptotic cells in nucleus pulposus of 15% BSHXF group decreased(P <0.01).6.Mitochondrial morphology and apoptotic bodies were observed by transmission electron microscope.Compared with the blank group,mitochondria in model group(TNF-αintervention)were swollen;Compared with the model group(TNF-α intervention),15%BSHXF group recovered the mitochondrial morphology and reduced the number of apoptotic bodies.7.The apoptosis rate of nucleus pulposus cells in model group was higher than that in control group(P<0.01).Compared with the model group(TNF-α intervention),the apoptosis rate of nucleus pulposus cells in 15% BSHXF group decreased(P<0.01).8.Compared with the control group,the content of reactive oxygen species(ROS)and the number of mitochondrial mitosis in model group were significantly increased(P <0.01).Compared with the model group(TNF-α intervention),the content of reactive oxygen species(ROS)and the number of mitotic mitochondria in the 15% BSHXF group decreased(P <0.01).9.SOD activity in nucleus pulposus was detected by Cu Zn/Mn-SOD method.Compared with the blank group,the SOD content and activity in model group(TNF-α intervention)were decreased(P<0.01).Compared with the model group(TNF-α intervention),the content and activity of SOD in the 15% BSHXF group were increased(P<0.01),and the ability of scavenging peroxide was enhanced.10.ATP content in model group was lower than that in control group(P<0.01).Compared with the model group(TNF-α intervention),the ATP content in 15% BSHXF group was higher(P<0.01).11.Compared with the blank group,there was no significant difference in AMPK(P>0.05),but the expression of p-AMPK protein was increased(P<0.01)in the model group(TNF-αintervention).Compared with the model group(TNF-α intervention),there was no significant difference in AMPK(P>0.05),but the expression level of p-AMPK was significantly increased(P<0.01)in the 15% BSHXF group.Compared with 15% BSHXF group,AMPK in AMPK inhibitor group had no significant difference(P>0.05),but the expression level of p-AMPK was decreased(P<0.01).Compared with SIRT-1 negative control group,the expression of SIRT-1 protein in SIRT-1 knockout group was decreased(P<0.01),while that in 15% BSHXF group was increased(P<0.01).Compared with 15%BSHX group,the expression of SIRT-1 protein in SIRT-1 knockout group decreased(P<0.01),and the expression of SIRT-1 protein in AMPK inhibitor group decreased(P<0.01).Compared with blank group,the expression of SIRT-1 protein in model group(TNF-αintervention)was increased(P<0.01).Compared with model group(TNF-α intervention),the expression of SIRT-1 protein in 15% BSHXF group was significantly higher(P<0.01).Compared with 15% BSHX group,the expression of SIRT-1 protein in AMPK inhibitor group was significantly decreased(P<0.01).Conclusion:This study reveals the mechanism of Bushen Huoxue Formula in treating intervertebral disc degeneration under the guidance of the theory of "Bushen Huoxue." In vitro cell experiments,it is found that the medicated serum of Bushen Huoxue Fang can inhibit the oxidative damage mediated by inflammatory factors,so as to treat the "stasis" of intervertebral disc microenvironment caused by ROS accumulation,regulate energy metabolism through AMPK/SIRT 1 signal pathway,promote the recovery of mitochondrial function,produce stable ATP,provide energy for normal cell function,further reduce nucleus pulposus cell apoptosis and delay intervertebral disc degeneration.This study provides the molecular biology research foundation for the treatment principle of Bushen Huoxue Formula and lays the foundation for the research of Chinese traditional medicine. |
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