Investigating the mechanism of Gubi Prescription against knee osteoarthritis based on PINK1/Parkin mitophagy pathway
Objective To observe the mechanism by which Gubi Prescription(GBF)regulates the PINK1/Parkin mitochondrial autophagy pathway against knee osteoarthritis(KOA).Methods Chondrocytes were randomly assigned to blank group,model group,positive drug group,and GBF group.Except for blank group,all other groups were induced with Carbonylcyanide-p-trifluoromethoxuphenylhydrazone(FCCP).The proliferation of chondrocytes was detected by CCK-8 method.The gene expressions of PINK1,Parkin,microtubule-associated protein 1 light chain 3(LC3),B cell lymphoma-2(Bcl-2),macromolecular B lymphoma(Bcl-xL),and myeloid cell leukemia-1(Mcl-1)were detected by RT-qPCR.A total of 60 mice were randomly separated into 4 groups:sham operation group,model group,positive drug group,and GBF group,15 mice each.Saffron O fast green staining was uesd to observe the cartilage tissue.The protein expressions ofPINK1,Parkin,LC3,Bcl-2,Bcl-xL,and Mcl-1 were detected by Western Blot.Results Compared with blank control group,the mRNA expressions of PINK1 and LC3 of model group were increased,while the OD value,mRNA expressions of Parkin,Bcl-2,Bcl-xL,and Mcl-1 were significantly decreased(P＜0.05);compared with model group,the mRNA expressions ofPINK1 and LC3 in positive drug group and GBF group were significantly decreased,while the OD value,Parkin,Bcl-2,Bcl-xL,and Mcl-1 mRNA were significantly increased(P＜0.05);compared with positive drug group,the mRNA expressions of PINK1 and LC3 in GBF group were significantly decreased,while the mRNA expressions of Parkin,Bcl-2,Bcl-xL,and Mcl-1 were significantly increased(P＜0.05);compared with sham operation group,the protein expressions ofPINK1 and LC3 Ⅱ/LC3 I in model group were significantly increased,while the protein expressions of Parkin,Bcl-2,Bcl-xL and Mcl-1 were significantly decreased(P＜0.05);compared with model group,the protein expressions of PINK1 and LC3 Ⅱ/LC3 I in positive drug group and GBF group were significantly decreased,while the protein expressions of Parkin,Bcl-2,Bcl-xL and Mcl-1 were significantly increased(P＜0.05);compared with positive drug group,the protein expressions ofPINK1 and LC3 Ⅱ/LC3 Ⅰ in GBF group were significantly decreased,while the protein expressions of Parkin,Bcl-2,Bcl-xL and Mcl-1 were significantly increased(P＜0.05).Conclusion GBF can delay cartilage degeneration,and its mechanism may be related to inhibiting the overexpression ofPINK1 and LC3,activating Parkin,promoting the increase of the expression of BCL2 family anti-apoptotic proteins Bcl-xL and Mcl-1,preventing the over-synthesis of PINKl/Parkin complex,and effectively reducing excessive mitochondrial autophagy.
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