Mechanism Of Bilobalide Regulating AMPK-SIRT3-mediated MQC Homeostasis To Improve Osteoarthritis

Osteoarthritis is the main disease that causes lameness in livestock,and its characteristics of cartilage degeneration and refractory characteristics force livestock to be eliminated in advance.Early osteoarthritis has no obvious clinical symptoms,and the golden period of treatment is often missed when it is diagnosed.Non-steroidal anti-inflammatory drugs commonly used in clinical practice can only relieve pain,but cannot reverse the disease.The development of safe and effective natural prevention and treatment drugs for livestock osteoarthritis is of great significance for improving the economic benefits of the industry,food safety and protecting the ecological environment.Bilobalide(BB)is the only sesquiterpene compound in Ginkgo biloba extract.It is believed that BB can inhibit cartilage degeneration,but the specific mechanism of action is not yet known.Mitochondrial quality control(MQC)system is a regulatory system of mitochondrial homeostasis,which maintains mitochondrial homeostasis through multidimensional integration of mitochondrial biogenesis,mitochondrial dynamics,mitophagy and mitochondrial redox biological processes.Imbalance in MQC homeostasis is an emerging mechanism of cartilage degeneration in osteoarthritis.Studies suggest that the AMPK-SIRT3 positive feedback loop can regulate the homeostasis of the mitochondrial MQC system,but its regulatory mechanism in BB protection of osteoarthritis remains unclear.This experiment is divided into three parts:(1)The first part: observe the protective effect of BB on rabbit osteoarthritis.The rabbit osteoarthritis model was established by intra-articular injection of 4% papain,and 80 mg/kg BB was continuously intervened for eight weeks.Micro-CT scanning and 3D reconstruction were used to observe the microstructure of subchondral bone and changes in related parameters;X-ray method was used to evaluate the joint space and osteophyte formation;the morphological changes of tibial and femoral cartilage were observed with naked eyes and Pelletier scoring was performed;HE staining and safranin O staining was used to evaluate the pathological changes of articular cartilage and OARSI score;Western blotting was used to detect ECM metabolic markers(type II collagen,ADAMTS4,MMP-3 and MMP-13),Nrf2 antioxidant system-related proteins(Nrf2,HO-1 and NQO1)and cyclooxygenase 2(COX-2);ELISA method to detect inflammatory factors(IL-1,IL-6,TNF-α and IL-15),cartilage and bone metabolic markers(COMP,C2C)in rabbit serum,CTX-II,TRACP-5b,β-CTx,PIINP and BALP)levels;micro-method detection to detect serum MDA,CAT and GSH levels;immunohistochemical method to detect AMPK-SIRT3 pathway-related proteins(AMPK and SIRT3),MQC Levels of system-associated proteins SOD2,PGC-1α,Drp1 and Parkin.To clarify the protective effect of BB on cartilage and subchondral bone damage in rabbit osteoarthritis.(2)The second part: BB regulates the mechanism of AMPK-SIRT3-mediated MQC homeostasis and inhibits cartilage degeneration.The ATDC5 chondrocyte inflammation model was established by using IL-1β,and the dosage of BB was screened by CCK-8 method;the proliferation of chondrocytes was detected by Ed U staining;the apoptosis level of chondrocytes was detected by Annexin V/PI double staining and TUNEL staining;ELISA,immunofluorescence Or Western blotting method to detect BB’s effect on chondrocyte ATP content,mitochondrial membrane potential(ΔΨm),ROS level and Nrf2 antioxidant system;Western blotting method to detect apoptosis-related proteins(Bcl2,BAX and Cleaved Caspase3)in chondrocytes,Mitochondrial biogenesis proteins(PGC-1a and NRF-1),mitochondrial fusion-related proteins(Mfn2 and OPA1),mitochondrial fission-related proteins(Drp1 and Fis1),mitochondrial redox proteins(SOD2)and mitophagy-related proteins(LC3II,P62,Parkin,PINK1 and VDAC1)levels;MDC fluorescence and transmission electron microscopy to observe the number and shape of chondrocyte mitophagosomes;Phagocytic flux;micro-method detection of MDA,CAT and GSH levels in chondrocytes.Then,the AMPK and SIRT3 pathway inhibitors Compound C and 3-TYP were used to establish a pathway blocking model,and the changes of key proteins in the downstream MQC system were detected to clarify the mechanism of action of the AMPK-SIRT3-mediated MQC system after BB intervened in chondrocytes.(3)The third part: the functional verification test of the protective effect of BB on rat osteoarthritis.Rat osteoarthritis model was established by ACLT,and 10 mg/kg BB was orally administered continuously for six weeks.The degree of joint pain in rats was detected by joint diameter detection,Von-Frey mechanical pain detection,cold sensitivity detection and knee joint pain vocalization;the changes of joint images were evaluated by Micro-CT scanning and X-ray methods;cartilage morphology observation and HE staining The pathological changes of articular cartilage were evaluated by staining with safranin O and ECM;the levels of ECM metabolic markers,Nrf2 antioxidant system-related proteins and COX-2 were detected by Western blotting;the levels of inflammatory factors,cartilage and bone metabolic markers in serum of rats were detected by ELISA;The level of MDA,CAT and GSH in serum was detected by micro-method;the level of related proteins of AMPK-SIRT3 pathway and MQC system was detected by immunohistochemical method.To verify the protective effect of BB on ACLT-induced osteoarthritis in rats.The results showed that:(1)In the papain-induced rabbit osteoarthritis model,BB inhibited the cartilage markers i NOS,ADAMTS4,MMP-3 and MMP-13 and the inflammatory factors IL-1,The levels of IL-6,TNF-α and IL-15 exert anti-inflammatory and anti-ECM degradation effects.BB up-regulates the Nrf2 antioxidant system to promote the secretion of CAT and GSH,and inhibits the level of MDA,thereby improving the body’s oxidative damage and pathological changes in cartilage.In addition,BB intervention can reverse the levels of cartilage and bone markers in rabbit osteoarthritis serum,inhibit the abnormal reconstruction of subchondral bone and the destruction of the three-dimensional structure of bone trabecula,indicating that BB has the effect of protecting cartilage and subchondral bone damage in rabbit osteoarthritis.(2)In vitro experiments,the ATDC5 chondrocyte inflammation model was established using IL-1β.BB was found to promote the expression of p-AMPK,SIRT3,PGC-1a,NRF-1,OPA1,Mfn2,SOD2,LC3 II,Parkin,PINK1 and VDAC1 in chondrocytes,inhibit the expression of Fis1,Drp1 and P62,and promote ATP synthesis and mitophagy Body formation,amelioration of IL-1β-induced abnormal mitochondrial membrane potential in ATDC5 chondrocytes.After pretreatment of chondrocytes with Compound C and 3-TYP,p-AMPK and SIRT3 proteins were significantly inhibited,and MQC system-related proteins PGC-1a,NRF-1,OPA1,Mfn2,SOD2,LC3 II,Parkin,PINK1 and VDAC1 expression was suppressed,whereas the expression of Fis1,Drp1 and P62 was significantly increased.BB can inhibit the expression of i NOS,ADAMTS4,MMP-3,MMP-13,BAX and Cleaved Caspase 3 proteins in chondrocytes induced by IL-1β,up-regulate the levels of Bcl2 and type II collagen,inhibit ROS levels,activate Nrf2 antioxidant system and inhibit cartilage Apoptosis.(3)It was verified in the rat osteoarthritis model established by ACLT,and it was found that after BB intervention,the degree of joint swelling of rats was reduced,and the pain of rats with osteoarthritis was inhibited.BB can promote the production of type II collagen in rat cartilage,inhibit the levels of COX-2,i NOS,ADAMTS4,MMP3,MMP13,IL-1β,IL-6,TNF-α and IL-15,and activate the Nrf2 antioxidant system to promote CAT and GSH expression,suppressed MDA levels,thereby improving oxidative damage and pathological features of cartilage in rats.In addition,BB intervention can reverse the levels of cartilage and bone markers in the serum of rats with osteoarthritis,and inhibit the damage of subchondral bone,indicating that BB has the effect of protecting rats from osteoarthritis.Provide data and research basis for the next step of BB clinical application in dairy cows,and provide a theoretical basis for the research of related target drugs.The above results show that:(1)BB alleviates cartilage and subchondral bone damage in rabbit osteoarthritis through anti-inflammation,anti-oxidation and anti-ECM degradation;(2)BB regulates PGC-1/NRF-1 pathway,Drp1 mitochondrial transfer,SOD2 Mitochondrial oxidation pathway and PINK1/Parkin pathway maintain MQC homeostasis in ATDC5 chondrocytes;(3)BB activates AMPK-SIRT3 positive feedback loop to mediate MQC inhibition of cartilage degeneration in osteoarthritis;(4)BB relieves osteoarthritis pain in rats,and play a protective role in rat osteoarthritis through anti-inflammatory,anti-oxidative and anti-ECM degradation effects.