Salvianolic Acid B Alleviates Intervertebral Disc Degeneration By Regulation Of Oxidative Stress In Vitro And In Vivo

Objective:Intervertebral disc degeneration(IDD)is one of the main causes of low back pain,and its etiology is complex and multi-factorial.Previous studies have shown that nucleus pulposus cell(NPC)damage caused by oxidative stress is one of the important factors in IDD progression.Salvia Miltiorrhiza has a wide range of pharmacological effects including treating cardiovascular diseases.Salvianolic acid B(SAB),the most abundant water-soluble compound in Danshen,has antioxidant properties,which can effectively eliminate the excessive production of reactive oxygen species(ROS)and inhibit the cell apoptosis induced by hydrogen peroxide(H2O2).Although SAB can regulate cell proliferation,apoptosis and cell function by inhibiting oxidative stress in many cell models,its effect on NPCs and intervertebral discs injured by oxidative stress and its underlying mechanism have not yet been reported.Our research intends to explore the effect and possible mechanism of SAB on rat IDD causeed by oxidative stress in in vitro and in vivo experiments.Methods:The nucleus pulposus tissues of the caudal intervertebral discs of adult male SD rats were isolated under aseptic conditions,and the primary NPCs were obtained,cultured and passaged.After oxidative stress was induced by adding 100μM H2O2 to the NPC culture medium in the drug group,SAB was added at a final concentration of lnM.The ROS content was explored by DCFH-DA method;the content of antioxidant indexes and inflammatory factors in each group were determined by ELISA method;the apoptosis rate of NPCs was determined by flow cytometry and Western blot;the mRNA and protein expression levels of collagen II(COL2),aggrecan(ACAN),MMP3,MMP13,ADAMTS4,and ADAMTS5 were investigated by Real time-PCR and Western blot;in addition,the JAK2,STAT3,p-JAK2 and p-STAT3 proteins were investigated by routine Western Blot method;the underlying mechanism of SAB alleviating IDD was verified by the application of JAK2/STAT3 pathway inhibitor AG490.In the in vivo experiment,the IDD model was induced by percutaneous acupuncture of the rat tail disc,and subsequently all rats in the SAB IDD group received SAB(20mg/kg)by oral gavage once per day.After 3 and 6 weeks,the tail vertebrae were scanned by X-ray and MRI for imaging analysis;after 6 weeks,the rats were sacrificed,and all disc specimens were taken for histomorphology,immunohistochemical staining,Western blot and biochemical analysis.Results:The in vitro experiments showed that H2O2 can successfully induce oxidative stress in NPCs,significantly increase the production of ROS and malondialdehyde(MDA),and inhibit the levels of antioxidant enzyme glutathione peroxidase(GSH-Px),catalase(CAT),superoxide dismutase(SOD),induce NPC apoptosis,increase the production of IL-1β,TNF-α and IL-6,decrease the expression of COL2 and AC AN,promote the expression of MMP3,MMP13,ADAMTS4,ADAMTS5,leading to the degeneration of NPCs.The addition of SAB can significantly inhibit oxidative stress,NPCs apoptosis,the secretion of inflammatory factors and the expression of matrix degrading enzymes,and alleviate the degeneration of NPCs.The addition of the pathway inhibitor AG490 significantly inhibited the protective effect of SAB on NPCs cells,proving that SAB exerts its efficacy through the JAK2/STAT3 signaling pathway.The in vivo experiments found that needle injury can decrease the space of the intervertebral disc,T2-weighed MRI signal intensity,and the water content of the intervertebral disc.Histological staining and semi-quantitatively analysis indicated that the nucleus pulposus tissue decreased,the arrangement of annular fibrosis was disordered,and the extracellular matrix was reduced,the intervertebral disc was significantly degenerated.Immunohistochemical staining analysis and Western blot found that the expression levels of COL2 and ACAN decreased,and the MMP3 increased.We also found that the expression levels of IL-1β and TNF-αsignificantly decreased.ELISA analysis showed that oxidative stress was significantly aggravated.However,the above IDD phenotype was significantly alleviated after SAB application in the experimental group.Conclusion:We have proved through in vitro and in vivo experiments that oxidative stress can induce NPC degeneration,and SAB can inhibit oxidative stress,cell apoptosis,inflammatory response and extracellular matrix degradation by activating the JAK2/STAT3 signaling pathway,thereby alleviating intervertebral disc degeneration,which may have broad application prospects in the medical treatment of IDD.