Effects And Mechanisms Of CHS On AMPK Signaling Pathway In IL-1? Induced Osteoarthritic Chondrocytes

Objective: OA is characterized by cartilage degradation,osteosclerosis,low-grade inflammation in the joints,etc.,which eventually causes joint inflammation and structural damage,thereby inducing cartilage destruction.Cartilage is responsible for maintaining a homeostasis between cytoplasm and metabolism.However,damage can impair chondrocyte function.Therefore,protecting the quality of chondrocytes and improving the function of chondrocytes have become effective means of treating OA.When cartilage is damaged,mitochondria will be damaged.Although the glycolytic capacity is increased,the number of mitochondria per cell is reduced,and the expression of PGC-1? in OA cartilage will be reduced.As a result,mitochondrial dysfunction can produce a large amount of ROS.Stress is closely related to tissue degenerative diseases such as OA,and its increased ROS can promote cartilage degeneration by damaging collagen and proteoglycans,and activating extracellular matrix metalloproteinases,and the activation of AMPK also affects the process of chondrocyte differentiation to a hypertrophic phenotype.Play a suppressive role.In addition,AMPK is generally considered to be a necessary regulator of cell energy homeostasis.AMPK phosphorylates PGC-1?,which further triggers SIRT1 to mediate PGC-1? acetylation.In chondrocytes,AMPK can inhibit oxidative stress and various inflammatory responses by activating PGC-1?.Chikusetsusaponin IV?(CHS)has many pharmacological properties.Although CHS is a good natural antioxidant and anti-inflammatory small molecule drug,it has not been studied in chondrocytes and osteoarthritis.This study aimed to investigate the effects of CHS on chondrocyte mitochondrial dysfunction,oxidative stress,and inflammation.IL-1? stimulates chondrocytes to mimic the pathogenesis of osteoarthritis.CHS can present dose-dependent maintenance of AMPK activity in damaged chondrocytes when stimulated by inflammatory factors.After completing the study of the precise role of CHS in protecting OA chondrocytes,AMPK / SIRT1 / is promoted by promoting mitochondrial biosynthesis The PGC-1? signaling pathway maintains mitochondrial function and oxidative stress in OA chondrocytes.The molecular mechanism of CHS in inhibiting the transition of OA chondrocytes to the hypertrophic phenotype also involves the AMPK / SIRT1 / PGC-1? pathway.Therefore,this study identified a new mechanism for the treatment of OA by CHS ? AMPK / SIRT1 / PGC-1? ? mitochondrial energy metabolism / hypertrophic phenotype ? chondrocyte function,providing important clinical application value for accurate understanding of bone and joint therapy and targeted therapy.Methods and Results:(1)CHS is a good natural anti-inflammatory small molecule drug,but it has not been studied in chondrocytes and osteoarthritis.Therefore,we first performed IL-1? and CHS toxicity tests on chondrocytes.Cell viability assay revealed that chondrocytes treated with 100 ng / ml IL-1? and 50 ?M CHS did not significantly reduce cell viability.Therefore,we used 100 ng / ml IL-1? and 50 ?M CHS in the following experiments.Chondrocyte in vitro models were prepared using IL-1? as OA stimulants,respectively.Based on the effect of CHS dose gradient on mouse primary chondrocyte viability,the optimal dose gradient and duration of CHS were selected.The results indicate that CHS treatment enhances the expression of AMPK / SIRT1 / PGC-1?.Based on the optimal dose and duration of CHS,the protein expression of AMPK?1 / 2,?1 / 2 and the level of AMPK?1 / 2 phosphorylation were measured.The results demonstrated that CHS protects the AMPK activity of OA chondrocytes.(2)Due to mitochondrial dysfunction,injury and synthesis defects in osteoarthritis chondrocytes,we examined the mitochondrial membrane potential of CHS-treated chondrocytes.Mitochondrial dysfunction was induced by IL-1? stimulation.Compared with the control group,IL-1? stimulation(Model group)for 24 hours can significantly reduce mito- chondrial membrane action potential.However,CHS treatment can significantly reduce IL-1?-inhibited mitochondrial dysfunction,which is manifested by mitochondrial membrane potential.The use of AMPK inhibitor Compound C(Cc)blocked the remission effect of CHS treatment.These results suggest that CHS may reduce IL-1?-induced chondrocyte mitochondrial dysfunction through the AMPK / SIRT1 / PGC-1? signaling pathway.(3)To investigate whether CHS can also reduce oxidative stress,we measured the formation of reactive oxygen species(ROS)in chondrocytes.The results indicate that IL-1? stimulation can increase ROS formation in chondrocytes.However,CHS treatment significantly reduced IL-1?-induced oxidative stress and significantly reduced ROS formation in chondrocytes.In addition,the use of Cc blocked the effect of CHS treatment.These results suggest that CHS may improve IL-1?-induced chondrocyte oxidative stress through the AMPK / SIRT1 / PGC-1? signaling pathway.(4)In order to determine the mechanism involved in the effect of CHS on mitochondrial function in the treatment of OA,the specific inhibitors of AMPK,SIRT1,PGC-1?,Cc,Ex527,and SR-18292 were added to the model group and CHS group to observe the effect of inhibitors on the pharmacological effects of CHS in order to clarify the signaling pathway of CHS effects.By detecting the expression of AMPK,SIRT1,PGC-1?,NF-?B p65 related proteins and their phosphorylated proteins.The results indicate that the protective effect and molecular mechanism of CHS on mitochondrial function of OA chondrocytes may play a role through the AMPK / SIRT1 / PGC-1? signaling pathway.(5)To further determine the function of CHS therapy,we tested the expression of extracellular matrix(ECM)synthesis and metabolism-related proteins in chondrocytes.The model group,and CHS group were treated with Cc,Ex527,and SR-18292,respectively.Western blot was used to detect the markers of normal and hypertrophic phenotypes of chondrocytes,Collagen II,Aggrecan,Collagen X,and MMP13.The results showed that compared with the non-stimulated control group,IL-1? stimulation increased the expression of MMP13 and Collagen X,while the expression of Aggrecan and Collagen II decreased.CHS treatment significantly reduced MMP13 and Collagen X in chondrocytes,and enhanced Aggrecan and Collagen II.The use of Cc,Ex527,and SR-18292 inhibitors blocked the treatment of CHS. These results suggest that CHS may improve IL-1?-induced chondrocyte ECM synthesis and metabolism through the AMPK / SIRT1 / PGC-1? signaling pathway.Conclusions:(1)CHS has a protective effect on AMPK activity in OA chondrocytes.(2)CHS relieves chondrocyte mitochondrial dysfunction through SIRT1 / AMPK / PGC-1? pathway,and alleviates IL-1?-induced oxidative stress in chondrocytes.(3)CHS maintains the balanced metabolism and breakdown of cartilage extracellular matrix through SIRT1 / AMPK / PGC-1? signaling pathway,inhibits the chondrocyte hypertrophy phenotype,and thereby improves cartilage function.