High Cholesterol Inhibits Tenogenic Differentiation In Tendon-derived Stem Cells Through ROS-activated NF-κB Signaling

Background and objectiveTendinopathy is a common soft tissue disease,characterized by chronic pain,swelling,or dysfunction.Although overuse is commonly considered as a major cause of tendinopathy,a large proportion of tendinopathy occurs in inactive individuals.Approximately 31%patient didn’t participate in strenuous exercise.Clinical studies have shown that high cholesterol is closely related to tendinopathy.Elevated serum cholesterol levels increased the risk of Achilles tendon injury in patients with hypercholesterolemia,and lowered the biomechanical properties of tendon,even delay the healing of tendon injury.In addition,cholesterol can also changed the phenotype of human tendon cells and reduced it’s collagen expression.Due to the limited understanding of the pathogenesis of high cholesterol-related tendinopathy,there is still a lack of effective treatment.Therefore,the deeply study of the cellular and molecular mechanisms in tendinopathy has important theoretical significance and application value.Tendon derived stem cells(TDSCs)have the potential for proliferation,cloning,and multifunctional differentiation,and can differentiate into tendon tissue in vitro or in vivo,as a potential cellular targets for studying the pathogenesis of tendinopathy.Abnormal differentiation of TDSCs is one of the important pathogenesis of tendinopathy.At present,there is no research report on the effect of cholesterol on TDSCs and the underlying mechanism.This study is to explore the effects of cholesterol on TDSCs and its underlying mechanisms,to help reveal the pathogenesis of high cholesterol-related tendinopathy,and to provide potential targets for the treatment of high cholesterol-related tendinopathy.Methods1.Cholesterol inhibits the differentiation of rat TDSCsFirst,we used cholesterol to stimulate TDSCs for 3 days,then we detected TNMD,Col 1 and other tendon-related gene expression by q-PCR to search the optimal concentration.Western blotting and cellular immunofluorescence staining techniques were also used to verify the result.2.Cholesterol inhibits tenogenic differentiation of TDSCs by up-regulating ROS levelsAfter TDSCs were stimulated by cholesterol,DCFH-DA probe was used to detect the changes of ROS levels in TDSCs by flow cytometry and inverted fluorescence microscopy.The total SOD activity detection kit and the MDA kit were used to detect the SOD activity and the change of MDA which can reflect the degree of oxidative stress damage.Western blotting was used to detect the expression of oxidative stress indicators CAT and NOX4 protein.Finally,the ROS scavenger NAC was used to verify whether it can reverse the up-regulation of CAT and NOX4 proteins and the inhibition of tenogenic differentiation of TDSCs by cholesterol.3.Cholesterol inhibits the differentiation of TDSCs by activating ROS-mediated NF-κB pathway ROS is linked to a number of signaling pathways,of which NF-κB is a key regulator as oxidative stress downstream.We examined the phosphorylation levels of IκBα and p65 proteins by Western blotting.The ROS scavenger NAC was used to verify whether it inhibits the cholesterol-induced phosphorylation of IκBα and p65.Finally,the NF-κB pathway inhibitor BAY was used to verify whether it can reverse the inhibitory effect of cholesterol on the tenogenic differentiation of TDSCs.4.Cholesterol induce tendon lesions through ROS activation of NF-κB signaling pathway In vivo10-month-old ApoE knockout mice fed a high fat diet was used as an animal model of hyperlipidemia.The histopathological changes of the Achilles tendon were analyzed by HE staining,and the tendon histology was scored using the Bonar scoring system.Immunohistochemical technique was used to detect the TNMD,Scx,oxidative stress indicator CAT,NOX4 protein and the expression of p-IκBα and p-p65 protein in NF-κB pathway.Results1.Cholesterol inhibits the tenogenic differentiation of rat TDSCs1 mg/dL,10 mg/dL,and 100 mg/dL cholesterol inhibited the expression of TNMD and Col 1 genes in a concentration-dependent manner.We used cholesterol with concentration of 10 mg/dL to stimulate TDSCs for 3 days in the following experiments.The expression of Scx,Col3,Egr,Fmod,Lum and Mkx genes was significantly decreased detected by q-PCR.in the expression of Scx,Col1,Col3 and TNMD proteins significantly decreased showed by Western blot and cellular immunofluorescence staining.The results indicated that cholesterol inhibits the tenogenic differentiation of rat TDSCs2.Cholesterol inhibits tenogenic differentiation of TDSCs by up-regulating ROS levelsAfter TDSCs were treated with Cholesterol for 3 days,ROS,MDA levels,and CAT and NOX4 protein expression were significanty up-regulated,but SOD activity was down-regulated.NAC can significantly lower cholesterol-induced ROS levels,and inhibits the CAT and NOX4 protein expression induced by cholesterol,reversing the inhibition of Sex,Coll,Col3 and TNMD protein expression by cholesterol.The results indicated that cholesterol inhibited the tenogenic differentiation of TDSCs by up-regulating ROS levels.3.Cholesterol inhibits the differentiation of TDSCs by activating ROS-mediated NF-κB pathwayAfter TDSCs were treated with 10 mg/dL cholesterol for 0,3,6,12,and 24h respectively,cholesterol was found to phosphorylate IκBα and p65 proteins in a time-dependent manner.The IKK inhibitor BAY can inhibit effectively cholesterol-induced phosphorylation of IκBα and p65.The ROS scavenger NAC also significantly inhibited cholesterol-induced phosphorylation of IκBα and p65.In addition,BAY reversed the inhibition of cholesterol on the expression of Scx,Col1,Col3 and TNMD proteins.The results indicated that cholesterol inhibited the tenogenic differentiation of TDSCs by activating ROS-mediated NF-κB pathway.4.Cholesterol induce tendon lesions through ROS activation of NF-κB signaling pathway In vivoBy HE staining,histologic results showed that the nucleus of the tendon cells became round,showing chondrocyte changes,and the red color stained by eosin was faded in the hypercholesterolemia group,suggesting that collagen degeneration occurred.These histological changes indicated that hypercholesterolemia caused tendon degeneration in mice Achilles tendon.Tendon tissue was scored using the Bonar scoring system.The mean Bonar score was significantly higher in the hypercholesterolemia group than in the control group(p<0.05).The results of immunohistochemistry showed that the expression of p-IκBα and p-p65 protein in the tenogenic marker TNMD,Scx,oxidative stress indicatror CAT,NOX4 protein and p-IκBα and p-p65 protein expression in NF-κB pathway were significantly decreased in the hypercholesterolemic group.The results suggested that high Cholesterol induce tendon lesions through ROS activation of NF-κB signaling pathway In vivo,as well.ConclusionTaken together,the results in this study suggeste that high cholesterol inhibite tenogenic differentiation of TDSC activated ROS-mediated NF-κB signaling pathway(as shown in the following figure),implying a pathogenesis of tendinopathy in hypercholesterolemia and suggesting a new mechanism underlying hypercholesterolemia-induced tendinopathy.This conclusion provides a theoretical basis for the use of statins to lower cholesterol levels and antioxidants for the treatment of hypercholesterolemia-associated tendinopathy.