Study On The Mechanism Of Asiatic Acid Alleviating Hypertrophy And Fibrosis Differentiation Of Articular Chondrocytes

Osteoarthritis(OA),the most common knee joint disorder,is characterized by a progressive degradation of articular cartilage.According to statistics,more than 80% of middle-aged and elderly people suffer from osteoarthritis of different degrees.The lack of effective clinical intervention means has seriously reduced people's quality of life and increased the economic burden of the society.However,the specific pathogenesis of OA is still unclear.Increasing evidence suggests that OA is closely associated with cartilage pathologies including chondrocyte hypertrophy and fibrosis.In the process of OA,hyaline cartilage differentiated into hypertrophic cartilage and fibrotic chondrocytes,inducing cartilage matrix degradation,endochondral vascular invasion and bone spurs formation.On the other hand,dedifferentiation of articular chondrocytes usually occurs during in vitro culture and expansion,which is characterized by the loss of COL-? and increase in type ? collagen(COL-?).The phenotypic instability and easy access to the fibroblast-like phenotype seriously affect the outcome of cartilage repair when using chondrocytes as seed cells.Taken together,how to inhibit chondrocyte hypertrophy and chondrocyte dedifferentiation is of great concern in both OA treatment and cartilage tissue engineering.Asiatic acid(AA),a pentacyclic triterpene isolated from Centella asiatica,has been reported to exhibit a variety of pharmacological effects,including antioxidant,anti-inflammatory,and hepatoprotective activities.Particularly,recent studies demonstrate that AA inhibits cardiac hypertrophy and liver fibrosis.However,whether AA could attenuate the hypertrophic differentiation or the fibrotic differentiation of articular chondrocytes has not been reported.We hypothesized that AA might attenuate chondrocyte hypertrophy or chondrocyte dedifferentiation.To verify this hypothesis,we designed the following experiment.Methods: Human OA chondrocytes were selected as the research object.After cartilagepieces were digested,cells were extracted for culture.Only cells at passage 1 were used in our study to avoid phenotype loss.1.First,the effects of AA(0,5,10,and 20 ?M)at different concentrations on the viability of chondrocytes were evaluated using a CCK-8 kit and a Live/Dead staining kit,and appropriate concentration was screened out.2.Human OA chondrocytes were divided into control group(control)and experimental group(AA).To observe the effects of AA on chondrocyte hypertrophy,immunofluorescence staining,qRT-PCR,and Western blot analysis were used to measure hypertrophic markers COL-X,MMP-13,and Runx2.At the same time,alkaline phosphatase staining is used to detect the expression of ALP.3.To observe the effects of AA on chondrocyte fibrosis,immunofluorescence staining,qRT-PCR,and western blot analysis were used to measure COL-? and ?-SMA.4.To observe the effects of AA on chondrocyte phenotypes,immunofluorescence staining,RT-PCR,and western blot analysis were used to measure SOX9,COL-?cartilage related factors and aggrecan(ACAN)expression changes.Meanwhile,the expression of ACAN was also detected by Alcian blue staining.5.In vitro,we further defined the molecular mechanism for the AA-mediated anti-hypertrophy and anti-fibrosis of human OA chondrocytes.6.Finally,we further verified the effect of AA on chondrocyte proliferation and fibrosis in an anterior cruciate ligament transection(ACLT)rat OA model by intra-articular injection with AA.Results: 1.From the results of CCK-8 and a Live/Dead staining,we found that Chondrocytes treated with 5 ?M AA showed comparable staining with untreated control,while 10 ?M and 20 ?M AA treatment induced more dead chondrocytes.Therefore,5?M AA was used for the subsequent experiments.2.We found that AA treatment significantly reduced the expression of chondrocyte hypertrophic factors.In human OA chondrocytes,AA treatment reduced expression of Runx2 and MMP-13 protein when compared to control group,as demonstrated by immunofluorescence staining.RT-PCR results showed that the mRNA level of Runx2 and COL10a1 in AA group was lower than that in control group,but with no statistical difference;the mRNA level of MMP-13 in AA group was significantly lower than that in control group(p < 0.01).Western blot results further revealed that,the protein level of Runx2,MMP-13,and COL-X in AA group were reduced in different degrees,as compared with control group.Additionally,a weaker staining of ALP after AA treatmentwas observed when compared to control group.The reduction in ALP further confirmed the anti-hypertrophic effect of AA.3.We found that AA inhibited key regulators of chondrocyte fibrosis.The staining of COL-? and ?-SMA in AA group was weaker compared with control group.COL1a1 mRNA level was significantly reduced by ~50% while ?-SMA m RNA was significantly reduced by ~22% in AA group compared with control group.Consistently,protein levels of COL-?and ?-SMA were also significantly inhibited by AA treatment.4.We further analyzed the effects of AA treatment on chondrocytic phenotype.It was showed that the immunofluorescence staining of ACAN,COL-?,and SOX9 in AA group was comparable with control group.Although gene expression of COL2a1,ACAN,and SOX9 in AA group was much higher than that in control group,the protein levels of these markers were similar between AA group and control group.In addition,the Alcian Blue staining showed no obvious difference between control group and AA group as well.5.We found AA treatment activated AMP-activated protein kinase(AMPK)and inhibited phosphoinositide-3 kinase/protein kinase B(PI3K/AKT)signaling pathway in vitro.6.The results of in vivo experiments showed that after 4 weeks and 8 weeks of AA injection,the ACLT group showed cartilage erosion,apparent hypocellularity,massive proteoglycan loss and a higher OARSI score compared with the ACLT+AA group.Moreover,histochemical analysis showed that,compared with the ACLT group,COL-? expression was increased while the expression of chondrocyte hypertrophy and fibrosis markers decreased in the ACLT+AA group,suggesting that AA slowed down chondrocyte hypertrophy and fibrosis during the development of osteoarthritis,consistent with our hypothesis.Conclusion: AA treatment could reduce hypertrophic and fibrotic differentiation,and maintain the chondrogenic phenotype of articular chondrocytes by targeting the AMPK/PI3K/AKT signaling pathway.Our study suggested that AA might be a prospective drug component that targets hypertrophic and fibrotic chondrocytes for OA treatment.