Protective Effect Of Shikonin On Vascular Endothelial Cell Injury Induced By H₂O₂ And Skin Microvascular Remodeling

[Objective]By exploring the protective effect of shikoin on vascular endothelial cells induced by H2O2 and the reconstruction of blood vessels in burn and scald wound tissue,this study laid a theoretical foundation for shikonin in traditional Chinese medicine treatment of burns and scald,which is of great significance for the clinical application of shikonin.[Method](1)The oxidative damage model of vascular endothelial cells stimulated by H2O2 was established.ELISA,flow cytometry,ROS fluorescence staining,MTT method,cellular immunofluorescence,Transwell,Western blot and other techniques were used.To observe the effects of shikonin on inflammation,apoptosis,ROS expression,cell viability,migration ability and endoplasmic reticulum stress-related proteins after oxidative injury,ER probe and ROS probe were used to label oxidative stress location.(2)A mouse model of second-degree burns on the ear and back skin was established,and the effects of shikonin on the expression of VEGF-A,VEGF-B and VEGF-C,MPO activity and vascularity in mouse-burned skin tissues were analyzed by using fluorescent staining,MPO activity assay and HE staining techniques.[Results](1)Vascular endothelial cells after H2O2 induction were treated with different concentrations of shikonin administration.The results showed that compared with the untreated group,the cell viability of the shikonin-treated group was significantly increased(P< 0.05),the content of inflammatory factors was significantly reduced(P< 0.05),the apoptosis rate was significantly decreased(P< 0.01),the positive expression of ROS was significantly reduced(P< 0.001),the TRX and8-OHd G were significantly reduced(P< 0.01),and the fluorescence intensity of ICAM-1 was significantly decreased(P< 0.01).(2)H2O2-induced vascular endothelial cells treated with different concentrations of shikonin administration showed that the cell migration ability was significantly enhanced in the shikonin-treated group compared with the untreated group(P< 0.001);the oxidative stress localization of vascular endothelial cells was found to be mainly concentrated in the ER;in addition,the expression of JAK2/STAT3 protein was increased in the shikonin-treated group compared with the untreated group(P<0.001).The expression of endoplasmic reticulum stress-related proteins(p-PERK,p-e IF2α,ATF4,CHOP)was significantly increased in endothelial cells after H2O2 induction compared with the blank control group(P< 0.001,P< 0.001,P< 0.001,P< 0.01),and the addition of shikonin treatment revealed that shikonin was able to down-regulate these endoplasmic reticulum stress-related proteins expression,and the expression of endoplasmic reticulum stress-related proteins(p-PERK,p-e IF2α,ATF4,CHOP)was significantly higher in the inhibitor WP1066+shikonin group than in the blank control group(P< 0.01,P< 0.01,P< 0.001,P<0.001).(3)The test of applying shikonin ointment to the burn mouse model showed that the fluorescence the intensity of VEGF-A and VEGF-C in the ear trauma of this treatment group was significantly higher than that of the burn model group(P< 0.001),while no statistically significant differences were seen in the expression of VEGF-B(P>0.05),MPO activity was significantly reduced(P< 0.001),and blood vessels in the dorsal trauma increased.[Conclusion](1)Shikonin can improve the inflammation and necrosis of vascular endothelial cells induced by H2O2;(2)Shikonin can enhance the migration ability of vascular endothelial cells induced by H2O2,and accumulate in the endoplasmic reticulum after entering endothelial cells.Shikonin may inhibit endoplasmic reticulum stress by indirectly regulating JAK2/STAT3 signal pathway,thus protecting endothelial cells;(3)Shikonin promotes angiogenesis in burned skin tissue,increases vascular density,and reduces neutrophil infiltration after burn injury.