Study On The Role Of Transient Receptor Potential Channel-Mediated Autophagy In Treating Osteoarthritis With Tetrahydrohyperforin

Backgroud and Objectives:Osteoarthritis?OA?is a common joint disease characterized by loss of articular cartilage and osteophyte formation.The degeneration of articular cartilage plays an important role in the pathogenesis of OA.It has been well-established that aging and apoptosis of chondrocytes contribute to cartilage degeneration.Therefore,maintaining the chondrocytes in a healthy condition appears to be an important factor for preservation of the entire cartilage and preventing its degeneration.Studies have shown that the occurrence of OA is associated with a decrease in the level of autophagy in cartilage tissue.Partial recovery of autophagy may help increase the viability of OA-derived chondrocyte.The transient receptor potential channel?TRPC?modulates various physiological activities through the regulation of Ca²⁺,including autophagy and apoptosis.Currently,research concerning the mechanism of TRPC-mediated autophagy is still at an early stage,and studies on the role of TRPCs in OA progression and articular cartilage degeneration remain rarely reported.Recent studies have reported that tetrahydrohyperforin?IDN5706?induces autophagy and thus exerts an anti-neurological degeneration effect.In addition,IDN5706 can improve spatial memory in mice with Alzheimer's disease,and its mechanism is related to the activation of TRPC3/6/7.At present,the role of IDN5706 in OA remains unclear.Therefore,this study aimed to investigate the effect of IDN5706 on the cartilage degeneration of OA,and to explore whether IDN5706 activates autophagy by regulating TRPCs,thereby exerting the effect of attenuating OA pathological injuryMethods:1.To explore in vivo whether IDN5706 ameliorates the degeneration of articular cartilage and affects autophagy in OA rats,we performed the following experiments:Following 1 week of acclimation,Sprague-Dawley?SD?rats were weighed and randomly divided into three groups:normal control group,OA model group,and IDN5706 group.Rats in the model group received intra-articular injection of collagenase solution in the right knee joint of the rat hindleg to construct a rat knee OA model.Rats in the normal control group were administered with an equal volume of saline as a control.The administration was performed twice,at days 1 and 4.Following two weeks,6g/kg IDN5706 or an equal volume of saline?OA group?was intragastrically administered once weekly for 6 weeks.The normal control group also received intragastrical administration of equal volume of saline once for 6 weeks.Afterwards,rats were then sacrificed and the articular cartilage tissues were separated from the knee joints in rats.Haematoxilin-Eosin?HE?staining and Safranin O staining were performed for histological evaluation.Histological changes of articular cartilage in three groups were evaluated using the Mankin scoring system.Quantitative real-time PCR?qRT-PCR?was used to detect the mRNA expression of matrix metalloproteinase-13?MMP-13?and vascular endothelial growth factor?VEGF?in cartilage tissues.Transmission electron microscopy was used to observe autophagosomes and autophagolysosome.Western blot was performed to detect the protein expression of mTOR,p-mTOR,Atg5,Beclin-1,and LC3-I and LC3-II.2.To examine in vitro whether IDN5706 can inhibit apoptosis and expression of OA-related genes in chondrocytes by activating autophagy,we performed the following experiments:Chondrocytes were isolated the knee joint of OA model rats and cultured with IDN5706?0,50,100,150 and 200?mol/L?for 24 h.Cell viability was assessed using a cell counting kit-8 assay.Afterwards,the concentration of 50?mol/L was selected for subsequent experiments.Chondrocytes isolated the knee joint of OA model rats?OA-derived chondrocytes?were cultured with IDN5706?50?mol/L?for 24 h.Next,the levels of inflammatory cytokines MMP-13 and IL-6 were detected with ELISA.Protein expression of p-mTOR,mTOR,Atg5,Beclin-1,LC3-I,LC3-II were measured by Western blot.OA-derived chondrocytes were pretreated with autophagy inhibitor3-methyladenine?3-MA?,then treated with IDN5706?50?mol/L?.Annexin V-FITC/PI flow cytometry was used to examine cell apoptosis.ELISA was used to examine levels of MMP-13 and IL-6 in OA-derived chondrocytes.3.To examine whether TRPC6-mediated autophagy is involved in DN5706-mediated inhibition of apoptosis and OA-related genes in OA chondrocytes in vitro and attenuation of pathological injury in OA rats,we performed the following experiments:The mRNA and protein expression of TRPC6 in cartilage tissues from normal and OA rats were examined by qRT-PCR and Western blot,respectively.OA-derived chondrocytes were transfected with si-TRPC6,then treated with IDN5706?50?mol/L?.Annexin V-FITC/PI flow cytometry was used to examine cell apoptosis in chondrocytes from OA rats.ELISA was used to examine levels of MMP-13 and IL-6in OA-derived chondrocytes.Protein expression of p-mTOR,mTOR,Atg5,Beclin-1,LC3-I,LC3-II were measured by Western blot.In vivo experiments:SD rats were weighed and randomly divided into three groups:normal control group,OA model group,IDN5706 group,IDN5706+SAR7334 group,and IDN5706+3-MA group.Rat OA model was established by intra-articular injection of type II collagenase.Following two weeks,6g/kg IDN5706 or an equal volume of saline?OA group?was intragastrically administered once weekly for 6 weeks.Rats in the IDN5706+TRPC6inhibitor SAR7334 group and IDN5706+autophagy inhibitor 3-MA group were injected with SAR7334 or 3-MA at the same time as IDN5706 injection.Afterwards,rats were then sacrificed and the articular cartilage tissues were separated from the knee joints in rats.HE staining and Safranin O staining were performed for histological evaluation.Histological changes of articular cartilage in three groups were evaluated using the Mankin scoring system.Western blot was performed to detect the protein expression of mTOR,p-mTOR,Atg5,Beclin-1,and LC3-I and LC3-II.Results:1.Effects of IDN5706 on articular cartilage degeneration and autophagy in cartilage of OA ratsHE staining and safranin O staining showed that IDN5706 reduced the pathological damage of OA and slowed down the cartilage degeneration of OA.In addition,IDN5706 treatment significantly reduced the highly expressed mRNA levels of MMP-13 and VEGF in articular cartilage tissue of OA rats.Transmission electron microscopy revealed that the normal rat articular cartilage cells were structurally intact.Decreased numbers of organelles,swelling mitochondria and vacuolar degeneration were observed in the cytoplasm of the OA group,and autophagosomes with double membranes were also decreased.Following treated with IDN5706,autophagosomes with double membranes were increased compared with the OA group.In addition,Western blot analysis showed that compared with the normal group,the protein levels of autophagy marker proteins?including LC3II,Beclin-1,and Atg5?in articular cartilage of OA rats were decreased,and the phosphorylation level of mTOR was increased.IDN5706 treatment resulted in a significant increase in protein levels of LC3-II,Beclin-1,and Atg5 in the articular cartilage of OA rats,and a significant decrease in mTOR phosphorylation state,suggesting that IDN5706upregulated the decreased autophagy in OA.2.Effects of IDN5706 on the apoptosis and levels of OA-related genes in chondrocytes and the underlying mechanism associated with autophagyCell viability in the five groups of OA-derived chondrocytes?0,50,100,150 and 200?mol/L IDN5706?exhibited no significant difference,indicating that there was no injury of IDN5706 for the chondrocytes.Thus,the concentration of 50?mol/L was chosen for the subsequent assays.The results from ELISA indicated that the levels of MMP-13 and IL-6 were significantly decreased in the IDN5706 group compared with the control group.Furthermore,IDN5706 treatment resulted in a marked decrease in phosphorylation of mTOR,and a significant increase in LC3-II/LC3-I ratio,protein levels of Beclin-1 and Atg5,in the IDN5706 group compared with the control group.Moreover,IDN5706 treatment significantly inhibited cell apoptosis.More importantly,inhibition of autophagy by 3-MA significantly attenuated the inhibitory effect of IDN5706 on OA-derived chondrocyte apoptosis and inflammatory cytokines.3.The role of TRPC6 and autophagy in the IDN5706-mediated inhibition of chondrocyte apoptosis and levels of OA-related genes,plus attenuation of pathological lesions in OA ratsTRPC6 mRNA and protein levels in cartilage tissue of OA mice were significantly lower than normal mice,and IDN5706 treatment significantly increased TRPC6mRNA and protein levels in cartilage tissue of OA mice.In vitro experiments demonstrated that knockdown of TRPC6 attenuated the inhibitory effect of IDN5706on chondrocyte apoptosis and OA-related genes?MMP-13 and IL-6?in OA-derived chondrocytes.TRPC6 kncokdown in OA-derived chondrocytes also significantly increased the phosphorylation of mTOR and decreased LC3-II/LC3-I ratio,and protein expression of Atg5,and Beclin-1,suggesting that TRPC6 knockdown inhibited autophagy in OA-derived chondrocytes.More importantly,TRPC6knockdown significantly attenuated the IDN5706-mediated upregulation of autophagy in OA-derived chondrocytes.In vivo experiments showed that compared with the IDN5706 group,both TRPC antagonist SAR7334 and autophagy inhibitor 3-MA treatment decreased the number of articular cartilage cells in OA rats,and increased the Mankin score.These results indicate that blocking of TRPC6 and autophagy attenuated the IDN5706-mediated attenuation of OA pathological lesions.In addition,SAR7334 treatment significantly reduced LC3-II/LC3-I ratio,the protein levels of Beclin-1 and Atg5 in articular cartilage of IDN5706-treated OA rats,and increased the phosphorylation of mTOR compared with the IDN5706 group,indicating that SAR7334 attenuatesd IDN5706-mediated upregulation of autophagy in cartilage tissue from OA rats.These findings indicated that TRPC6 involved in the upregulation of autophagy by IDN5706.Conclusion:1.IDN5706 prevented articular cartilage degeneration and affected autophagy in OA rats;2.IDN5706 inhibited the apoptosis and OA-related genes MMP-13 and IL-6 in OA-derived chondrocytes by activating autophagy;3.IDN5706 inhibited the apoptosis and OA-related genes in OA-derived chondrocytes and attenuated the pathological injury in OA rats by upregulating TRPC6-mediated autophagy.