The Molecular Mechanism Of The Effect Of Oleanolic Acid On Treating Hypertrophic Scar

Hypertrophic scar is the body’s wound healing process of a product, whose main ingredient is fibrin, and the overgrowth of fibrin could induce the formation of hypertrophic scars. In China, there are91.4%burn patients and44.6%surgical patients in the rehabilitation phase of hypertrophic scar formation, and an annual increase of about32.75million hypertrophic scar patients[1,2]. Therefore, exploring the mechanism of hypertrophic scars and finding safe and effective natural medicine should be to execute imminently.Hypertrophic scars skin tissue was provided by Shanghai Changhai Hospital burn department, tissue culture method using cultured fibroblasts of the skin, giving oleanolic acid treatment, detected the follow effect of OA:(1) Human hypertrophic scar fibroblasts apoptosis detection:cell viability was detected by MTT; The effect of OA on HSFBs Morphology after treatment with OA for12h; Hoechst33258staining apoptotic DNA fragmentation was measured.(2) Detecting to the expression of apoptosis-related proteins and genes:The protein expression and phosphorylation of ERK1/2、JNK and p38were detected by western blot, FQ-PCR detected mRNA expression of apoptosis-related gene, such as caspase-3, caspase-8, caspase-9and Apaf-1.(3) Study on the signal transduction pathway:after treatment with p38MAPK and JNK signaling pathway specific inhibitor SB203580and SP600125for blocking the signal pathway respectively, oleanolic acid had an effect on hypertrophic scars fibroblasts via observing the expression of ERK1/2, JNK, p38and their phosphorylation.(4) The effect of OA on mitochondrial translocation of mitochondrial apoptotic proteins, such as Bad, Bax, Cyt-c and AIF, and the total mitochondrial proteins Bcl-2, Bax and Bid.(5) The effect of OA on hypertrophic scar of rabbit ears:Via the model of hypertrophic scar of rabbit ears, we detected the mRNA expression of MMP-1, MMP-2, TIMP-1, caspase-3, caspase-9, P311and TGF-β1, SEI and the dense of collagen fiber.The results showed that (1) After treatment with0,2.5,5,10,20,40,80μg/ml oleanolic acid for12h,24h and48h, human hypertrophic scar fibroblasts viability was inhibited remarkably in a dose-and time-dependent manner; HSFBs Morphology after treatment with OA for12h became shorter, and what’s more, became round, the volume of HSFBs became small, and the outline of HSFBs became twilight; After treatment with10,20,40μg/mL oleanolic acid for12h, apoptotic DNA fragmentation was measured via Hoechst33258staining, the detection indicated that oleanolic acid could promote the formation of asymmetric cell state, then DNA fragmentation, and further forming withered dead bodies.(2) Human hypertrophic scar fibroblasts starvation for6h (Cultured with2%FBS for6h), after treatment with0,20,40μg/ml oleanolic acid for1h, western blotting detect ERK1/2, JNK, p38, and their phosphorylation, phosphorylation of JNK and p38were remarkably increased, phosphorylation of ERK1/2was no difference among these groups; treatment with0,10,20,40μg/ml oleanolic acid for6h, expression of caspase-3/9mRNA and Apaf-1mRNA were remarkably increased in a dose-dependent manner.(3) Human hypertrophic scar fibroblasts starvation for6h, pretreatment with20μM SB203580or SP600125for1h, then treatment with20,40μg/ml oleanolic acid for1h, the results showed that midiation of the protein phosphorylation was significantly change via blocking the signaling pathways associated with oleanolic acid-related pathways.(4) Treatment with oleanolic acid10,20,40μg/ml for12h, a significant reduction in mitochondrial cytochrome c (Cyt-c), apoptosis-inducing factor(AIF) content and Bad, cytosolic Cyt-c and apoptosis-inducing factor (AIF) increased significantly, Bad only had the increasing tendency but no significant difference. Oleanolic acid significantly increased expression of Bax protein and significantly reduced Bcl-2protein expression, leading Bax/Bcl-2ratio rising. Treatment with10,20,40μg/ml oleanolic acid for6h, oleanolic acid significantly reduced expression of cytoplasmic Bax protein, and significantly increased expression of mitochondrial Bax protein.(5) In the model of hypertrophic scar of rabbit ears, OA could reduced the mRNA expression of MMP-1, TIMP-1, P311and TGF-β1, promoted the mRNA expression of MMP-2, caspase-3and caspase-9, reduced the amount of collagen cell, inhibited SEI, and promoted collagen fiber sedimentation.In summary, oleanolic acid significantly inhibited human hypertrophic scar fibroblasts growth, activated p38and JNK phosphorylation of proteins, but did not affected the phosphorylation of ERK proteins. Oleanolic. acid did not antagonize the p38pathway inhibitor (SB203580) p38inhibition, but enhanced phosphorylation of JNK. JNK pathway was suppressed after treating with JNK signal inhibitor (SP600125), but OA could promote the phosphorylation of p38protein. Oleanolic acid promoted Bax translocation, mitochondrial cytochrome C release and apoptosis-inducing factor AIF, the expression of Bax and caspase-3 protein and the ratio of Bax/Bcl-2, reducing the expression of Bcl-2proteins promote Apaf1and capase-3,-8,-9mRNA expression. In the model of hypertrophic scar of rabbit ears, OA could reduced the mRNA expression of MMP-1, TIMP-1, P311and TGF-β1, promoted the mRNA expression of MMP-2, caspase-3and caspase-9, reduced the amount of collagen cell, inhibited SEI, and promoted collagen fiber sedimentation. OA by activating p38and JNK pathways and mitochondrial death pathway to induce apoptosis, is a better treatment of hypertrophic scars natural medicine.