The Effect Of Haoqin Huaban Recipe On The Proliferation, Oxidative Damage And Inflammation Of HaCaT Cells Induced By UVB

Photosensitive dermatosis is caused by ultraviolet radiation.It is a common and multiple dermatosis with erythema,edema or multiform skin lesions,conscious burning and itching in the sun exposed area of the skin.Traditional Chinese Medicine calls it "Rishaichuang".The damage of skin keratinocytes caused by ultraviolet radiation is a key part of the pathogenesis of photosensitive skin diseases.Haoqinhuabanfang,which contains 8 Chinese herbal medicines such as Qinghao,Qinjiao,Danpi,Chishao,etc.,has the effect of clearing heat,cooling blood and detoxifying,and has good curative effect in clinical application.At present,there is no basic research at home and abroad to protect keratinocytes from ultraviolet damage.In this study,a pathological model of human immortalized epithelial keratinocytes(HaCaT cells)injured by mid-wave ultraviolet radiation was established.The purpose is to simulate human immortalized epithelial keratinocytes damaged by ultraviolet B(UVB)radiation,to explore whether Haoqinhuabanfang has protective effects on the damage of HaCaT cells induced by UVB,and to explore the possible mechanism of action,enriching the scientific connotation of method of clearing heat and cooling blood for detoxification in the treatment of photosensitive skin diseases.Research objectives:To establish the pathological model of HaCaT cells damaged by UVB radiation,and to explore the protective mechanism of Haoqinhuapan on the photodamage of HaCaT cells,so as to provide scientific basis for the effective treatment of photosensitive dermatosis with the method of "clearing heat,cooling blood and detoxifying".Research methods:1.Cell cultureHaCaT cells were cultured in 1640 medium containing 10%fetal bovine serum.When the cell density increased to more than 80%,the cells were collected and quantitatively seeded in a petri dish or 96-well cell culture plate.2.Determination of cytotoxicityHaCaT cells were treated with different doses of Haoqinhuabanfang medium for a certain period of time,MTT assay was used to detect cell proliferation,the inhibition rate of different doses of drugs on HaCaT cells was calculated,and the dose with the cell inhibition rate of less than 10%was selected as the maximum non-toxic dose.3.UVB irradiation and groupingAccording to the maximum non-toxic concentration,we determined the three different doses of Haoqinhuabanfang.According to the experimental design,HaCaT cells were irradiated with 300 MJ/cm2UVB,and then treated with high,middle and low doses of Haoqinhuabanfang.Normal cultured cells were used as blank control group,and UVB irradiated cells were used as UVB model group without drug intervention.4.Effect of Haoqinhuabanfang on proliferation of HaCaT cellsMTT method was used to detect cell proliferation.5.Effect of Haoqinhuabanfang on oxidative damage of HaCaT cellsThe activity of superoxide dismutase(SOD),catalase(CAT)and the content of malondialdehyde(MDA)were determined by absorbance method.6.Effect of Haoqinhuabanfang on inflammation of HaCaT cellsELISA method was used to detect endothelial cell-leukocyte adhesion molecule-1(ELAM-1),intercellular adhesion molecule-1(ICAM-1),vascular intercellular adhesion molecule-1(VCAM-1),and interleukin-10(IL-10),the content of transforming biological factor ?(TGF-?).Real Time PCR method was used to detect the relative expression levels of ELAM-1,ICAM-1,VCAM-1,IL-10 and TGF-?.Research Result:1.Cytotoxicity determination and cell groupingThe measurement of cytotoxicity found that the 1.25%Chinese medicine had the lowest inhibition rate on cell activity,and the corresponding Chinese medicine concentration was 12.1 mg/mL,so the maximum non-toxic dose of Chinese medicine could be determined.The experimental cells were divided into five groups:blank group,UVB model group,Haoqin Huabanfang high-dose group(Chinese medicine concentration is 12.1 mg/mL),Haoqin Huabanfang medium-dose group(Chinese medicine concentration is 6.05 mg/mL),Haoqinhuabanfang low-dose group(Chinese medicine concentration is 3.03mg/mL).2.Effect of Haoqinhuabanfang on proliferation of HaCaT cellsCompared with the blank group,the proliferation activity of HaCaT cells in the UVB model group was significantly reduced,and the difference was statistically significant(P<0.01).Compared with the UVB model group,HaCaT cell proliferation activity was significantly enhanced in the high,medium and low dose groups of Haoqinhuafangfang,the difference was statistically significant(P<0.01),indicating that the decrease in cell proliferation activity caused by UVB radiation,and high,medium and low doses of Haoqinhuabanfang can improve cell proliferation activity and reverse it to a certain extent.3.Effect of Haoqinhuabanfang on oxidative damage of HaCaT cellsCompared with the blank control group,the expression of SOD in the UVB model group decreased(P<0.05);compared with the UVB model group,the SOD expression in the high-dose group of Haoqinhuabanfang increased(P<0.05),suggesting that high-dose Haoqinhuabanfang could enhance SOD activity.Compared with the blank control group,the amount of CAT required to decompose 1 ? mol of H2O2 in the UVB model group increased,suggesting that CAT activity was significantly reduced(P<0.01);compared with the UVB model group,the amount of CAT required to decompose 1 ? mol of H2O2 in high-dose group of Haoqinhuabanfang decreased,indicating that CAT activity was enhanced after intervention of high-dose Haoqinhuabanfang(P<0.05).Compared with the blank control group,the MDA content of the UVB model group was significantly increased(P<0.01);compared with the UVB model group,the MDA content of the high-dose group of Artemisia Qinhuabanfang was significantly decreased(P<0.01),showing the positive effect of high-dose Haoquabanfang on MDA.It shows that high-dose Haoqinhuabanfang can improve the oxidative damage caused by UVB to a certain extent.4.Effect of Haoqinhuabanfang on inflammation of HaCaT cellsThe detection of inflammation-related factors by ELISA showed that compared with the blank control group,the levels of IC AM-1,VC AM-1,EL AM-1,and IL-10 in the UVB model group all increased(P<0.05),and the expression of TGF-? content decreased(P<0.05),indicating that UVB irradiation can induce inflammation;compared with the UVB model group,the contents of ICAM-1,VCAM-1,ELAM-1,and IL-10 in the high-dose group of Haoqinhuabanfang were all reduced(P<0.05),the content of TGF-? increased(P<0.05),indicating that high-dose Haoqin Huabanfang can effectively reduce the inflammation induced by UVB irradiation.The results of RT-PCR showed that compared with the blank group,the gene transcription levels of IC AM-1,VC AM-1,EL AM-1 and IL-10 in the UVB model group increased,and the level of TGF-? gene transcription decreased(P<0.05);Compared with the model group,the relative expression of ICAM-1 and IL-10 genes in the high,medium and low dose groups of Haoqinhuabanfang were reduced(P<0.05),and the VCAM-1 and The relative expression of ELAM-1 gene was reduced(P<0.05),and the relative expression of TGF-? gene was increased in the low-dose group of Haoqinhuabanfang(P<0.05).Conclusion:UVB radiation can decrease the proliferation of HaCaT cells and induce oxidative damage and inflammation.The Haoqinhuabanfang can inhibit UVB induced oxidative damage of HaCaT cells by enhancing the activities of SOD and CAT and reducing the production of Lipid peroxidation MDA,it can inhibit the UVB-induced inflammation of HaCaT cells by reducing the expression of ICAM-1,VCAM-1,ELAM-1 and IL-10 in HaCaT cells,repair skin barrier function by enhancing cell proliferation activity and up-regulating the expression of fibrogenic factor TGF-?.The above three aspects can protect HaCaT cells from photodamage induced by UVB.Haoqinhuabanfang was established on the basis of the theory of "Phototoxic stagnant skin" with the core treatment of "clearing heat and cooling blood and detoxifying".The method of clearing heat and cooling blood and detoxifying,which is summarized by traditional Chinese medicine,is an effective method to prevent and treat photosensitive skin diseases,this study proves the reliability and scientific basis of the theory.