Effects Of Ultrasound-targeted Simvastatin Microbubbles On Reverse Cholesterol Transport In Rabbit Knee OA Chondrocytes

Part ? Effect of ultrasound-targeted simvastatin microbubbles on chondrocytes metabolism of rabbit with knee osteoarthritisObjective:To investigate the effect of ultrasound-targeted simvastatin(SV)microbubbles on chondrocytes metabolism of rabbit with knee osteoarthritis(OA).Methods:(1)The cultivation chondrocytes of rabbit.Sixteen healthy New Zealand adult male rabbits were selected(2-2.5kg),of which tweleve underwent knee anterior cruciate ligament transection(ACLT),and the early knee OA model was established 4weeks later.Normal and OA rabbit right knee chondrocytes were extracted by enzyme digestion and cultured in vitro.OA chondrocytes were identified by immunofluorescence staining.(2)Different ultrasound intensity intervention:naked microbubbles were added to OA cartilage cell culture medium.Continuous ultrasonic intervention with frequency 1MHz,ultrasonic intensity 0W/cm~2(control group),0.3W/cm~2,0.6W/cm~2,1.0W/cm~2for 30s,quantitative real time PCR(Polymerase Chain Reaction,PCR)was used to detect the expression of proteoglycan and type ? collagen mRNA in OA chondrocytes.(3)Ultrasound targeting different concentrations of simvastatin microbubbles:after the simvastatin microbubbles were diluted to the concentrations of 1uM,10uM and 100uM,0.1%dimethyl sulfoxide(dimethylsulfoxide,DMSO)was used as the control group.Microbubble rupture was induced by 1MHz and 0.3W/cm~2ultrasound targeting.The mRNA expression of proteoglycan and type ? collagen in OA chondrocytes was detected by quantitative real time PCR.Results:(1)Compared with the normal group,the expression of proteoglycan in OA chondrocytes was significantly decreased(P<0.01).(2)Compared with the 0 W/cm~2,the expression of proteoglycan and type ? collagen in the 0.3 W/cm~2group was significantly increased(P<0.05).Compared with the 0.3W/cm~2,the proteoglycan and type ? collagen mRNA expression in the 1.0 W/cm~2group were significantly decreased(~~aP<0.01).(3)Compared with 0.1%DMSO group,1uM simvastatin group of type ? collagen and proteoglycan expression level increased significantly(P<0.001).Compared with 1uM simvastatin group,type ? collagen and proteoglycan expression level in the 10uM and 100 uM simvastatin group were decreased significantly(P<0.01).Conclusion:(1)The level of proteoglycan in OA chondrocytes was decreased,which can be used as a key index to distinguish normal chondrocytes from OA chondrocytes.(2)Ultrasound-targeted simvastatin microbubbles promoted the expression of proteoglycan and type ? collagen in OA chondrocytes.Part ? Effect of ultrasound-targeted simvastatin microbubbles on reverse cholesterol transport in chondrocytes of rabbit with knee osteoarthritisObjective: To investigate the effect of ultrasound-targeted SV microbubbles on reverse cholesterol transport(RCT)in chondrocytes of rabbit with knee osteoarthritis.Methods: Twenty healthy New Zealand adult male rabbits were selected(2-2.5kg),of which fifteen of them received right knee anterior cruciate ligament transection(ACLT).After 4 weeks,normal and OA chondrocytes were cultured and identified in vitro.The chondrocytes were divided into the Normal control group(N),OA group(OA),ultrasound-mediated microbubbles group(UM),Pure ultrasound group(PU),Pure SV group(PSV)and ultrasound-mediated SV microbubbles group(USV).All groups were intervened once every 48 hours.Detection index of chondrocyte metabolism and lipid metabolism: The extracellular matrix of product(type ? collagen and proteoglycan),Peroxisome proliferator activated receptor(PPAR ?)and Matrix metalloproteinase-13(MMP-13)expression levels were detected by Western blot(WB)in each group.Cholesterol efflux was measured by 25-NBD cholesterol labelling experiment.And triglycerides(TG)levels was analyzed by oil red O staining method.The total cholesterol and high density lipoprotein cholesterol(HDL-C)level were measured by enzyme analysis method.Results:(1)Compared with normal group,the expression levels of proteoglycan,type ? collagen and PPAR ? in OA group were significantly decreased,but the expression of MMP-13 were significantly increased(a P <0.01).Compared with OA group,the expression levels of proteoglycan and type ? collagen in PU,PSV and USV groups were significantly increased,the expression of PPAR ? in PSV and USV groups were significantly increased,while the expression levels of MMP-13 were significantly decreased(P < 0.05).Compared with PU group,the expression of proteoglycan,type ? collagen and PPAR ? in USV group were significantly increased,while MMP-13 level were significantly decreased(b P <0.05).Compared with PSV group,the expression level of proteoglycan,type ? collagen and PPAR ? in USV group were significantly increased,while MMP-13 level were significantly decreased(c P < 0.05).(2)Compared with normal group,Cholesterol efflux rate was significantly decreased,while TG levels,the total cholesterol and TG/ HDL-C ratio were significantly increased in OA group(a P < 0.001).Compared with OA group,Cholesterol efflux rate was significantly increased,while TG levels,the total cholesterol and TG/ HDL-C ratio were significantly decreased in PSV and USV groups(P < 0.001).Compared with PSV group,Cholesterol efflux rate was significantly increased,while TG levels,the total cholesterol and TG/ HDL-C ratio were significantly decreased in USV group(~bP <0.05).Conclusion: Ultrasound-targeted SV microbubbles promoted the metabolism and lipid metabolism of rabbit knee OA chondrocytes.The mechanism of promoting synthetic metabolism and inhibiting catabolism of chondrocytes might be closely related to activating PPAR ? and increasing the rate of intracellular cholesterol outflow.