Study On The Mechanism Of Qingxuedu Capsule Treating Psoriasis Vulgaris Based On Sonic Hedgehog Signal Pathway

[Objective]1.To explore the mechanism of Qingxuedu capsule treating psoriasis vulgaris by observing the changes of Symptoms,pathological manifestations of skin lesions,proliferation rate of keratinocytes,apoptosis index and expression of related inflammatory factors in psoriatic mice treated with Qingxuedu capsule.2.To explore the mechanism of Qingxuedu capsule in the treatment of psoriasis vulgaris by observing the changes of HaCaT cell proliferation rate,apoptosis index and sonic hedgehog(Shh)signal pathway expression after Qingxuedu capsule stimulated HaCaT cells.3.By observing the changes of proliferation rate,apoptosis index and anti apoptotic protein Bcl-2 expression of HaCaT cells stimulated by Qingxuedu capsule and rhshh;Verify that Qingxuedu capsule can promote HaCaT cell apoptosis and inhibit HaCaT cell proliferation by inhibiting Shh signal pathway and Bcl-2 expression.[Method]48 SPF BALB/c mice were randomly divided into 6 groups: blank group,methotrexate group,Qingxuedu capsule low,medium and high dose group.There were 8 mice in each group.The mouse model of psoriasis vulgaris was prepared with 5% imiquimod external liniment.While imiquimod prepared the psoriasis model,each group of mice were treated with corresponding drugs.Mice in each group were treated for 7 days.The researchers observed and recorded the skin lesion color,scale degree and skin thickness of mouse in each group every day,and then scored the Psoriasis Area and Severity Index(PASI).After the intervention,all mice were killed at one time,and the skin tissue was taken to detect the relevant indexes.The pathological changes of skin tissue was detected by HE staining and scored by Baker method.The expression rate of Ki-67 positive cells in mouse skin tissue was detected by immunohistochemical staining.The apoptosis index(AI)of keratinocytes in mouse skin tissue was detected by terminal dexynucleotidyl teanferase(Td T)mediated d UTP nick end labeling(TUNEL).The protein expression of Bcl-2 in mouse skin tissue was detected by Western blot.The m RNA expression of Bcl-2 in mouse skin tissue was detected by RT-PCR.And the protein contents of Interleukin-1 beta(IL-1β)and Interleukin-6(IL-6)in mouse skin tissue were detected by enzyme linked immunosorbent assay(ELISA).The second part is HaCaT cell experiment.Seven groups were set up:blank group,control group and Qingxuedu capsule 0.125、0.25、0.5、1、and 2 mg/m L dose group.The blank group was added with 200μl complete medium,and the other groups were added with 200μL cell culture medium in good growth condition,5 wells in each group,cultured for 24 hours.Then give the corresponding concentration of Qingxuedu capsule solution to stimulate for 24 hours.After the intervention,the cell survival rate was detected by CCK8.According to the effect of drugs on cell survival rate and cell morphology,the appropriate drug concentration of Qingxuedu capsule was selected for subsequent experiments.The formal experiment was divided into four groups: the control group,Qingxuedu capsule low dose group,Qingxuedu capsule medium and high dose groups.The cells in good growth condition were inoculated into 6-well plates.Each group is provided with 5 repeat wells and culture for 24 hours after inoculation.Then give the corresponding concentration of Qingxuedu capsule solution for intervention.After the intervention technology,the apoptosis of HaCaT cells was detected by flow cytometry,and the effect of Qingxuedu Capsule on the apoptosis rate of HaCaT cells was analyzed.Repeat the above cell culture and intervention process,the relative m RNA expression of Shh,SMO,Gli1 and Bcl-2 in HaCaT cells was detected by RT-PCR,and to analyze the effect of Qingxuedu capsule on the m RNA expression of Shh,SMO,Gli1 and Bcl-2 in HaCaT cells;Repeat the above cell culture and intervention process,the protein expression of Shh,SMO,Gli1 and Bcl-2 in HaCaT cells was detected by western blot,and the effect of Qingxuedu capsule on the protein expression of Shh,SMO,Gli1 and Bcl-2in HaCaT cells was analyzedIn the third part,HaCaT cells were cultured routinely.HaCaT cells with good growth were divided into control group,rhshh induction group(0.5 μg/ml),rhshh induction group(0.5 μg/ml)+ Qingxuedu capsule group and rhshh induction group(0.25 μg/ml)+ Qingxuedu capsule group,5 wells in each group,cultured for 24 hours;then Qingxuedu capsule and rhshh protein were given to intervene HaCaT cells for 24 hours;after the intervention,the cell proliferation was detected by CCK8,and the effect of Qingxuedu Capsule on the proliferation of HaCaT cells induced by rhshh was analyzed.Repeat the above cell culture and intervention process,the apoptosis rate was detected by flow cytometry,and the effect of Qingxuedu capsule on HaCaT cell apoptosis induced by rhshh was analyzed;Repeat the above cell culture and intervention process,the expression of Bcl-2protein was detected by western blot,and the effect of Qingxuedu capsule on Bcl-2 protein of HaCaT cells induced by rhshh was analyzed;repeat the above cell culture and intervention process,the relative m RNA expression of Bcl-2 was detected by RT-PCR,and then the effect of Qingxuedu Capsule on Bcl-2 m RNA of HaCaT cells induced by rhshh was analyzed.[Result]1.Compared with the blank group,the back skin erythema and the symptoms of scale and skin thickening increased significantly in model group(P<0.0001);the score of baker method increased significantly in model group(P<0.0001);the rate of Ki-67 positive cells in epidermis increased significantly in model group(P<0.0001);the index of apoptosis increased significantly in model group(P<0.05);the expression of Bcl-2protein increased significantly in model group(P<0.05,P<0.01);the relative expression of Bcl-2 m RNA increased significantly in model group(P<0.05,P<0.0001,P<0.001);IL-1 β And IL-6 increased significantly in model group(P<0.01).Compared with the model group,on the 7th day,the symptoms of mice in the methotrexate group,Qingxuedu capsule medium and high dose groups were significantly alleviated,and PASI score decreased significantly(P<0.0001);the rate of Ki-67 positive cells decreased significantly in methotrexate group,Qingxuedu capsule medium and high dose group capsule(P<0.01,P<0.0001);apoptosis index decreased significantly in methotrexate group,Qingxuedu capsule medium and high dose group(P<0.05);the content of Bcl-2 protein decreased significantly in the methotrexate group,Qingxuedu capsule medium and high dose group(P<0.05,P<0.01,P<0.001);and the relative expression of Bcl-2m RNA decreased significantly in the methotrexate group and Qingxuedu capsule low dose,medium and high dose group(P<0.01,P<0.001,P<0.0001);the content of IL-6 decreased significantly in methotrexate group,Qingxuedu capsule medium and high dose group(P<0.05,P<0.01),the content of IL-1 β decreased significantly in methotrexate group and Qingxuedu capsule high dose group(P<0.01).2.The results of CCK8 showed that the inhibitory effect of Qingxuedu capsule on HaCaT cells increased with the increase of concentration and time;after stimulate with Qingxuedu capsule for 24 hours,the concentration below 0.25 mg/ml had no obvious cell inhibition,and the concentration below 0.125 mg/ml had no obvious cell inhibition after 48 hours.The half maximal inhibitory concentration(IC50)of Qingxuedu capsule for 24 hours was 1.859 mg / ml;The IC50 was 0.66mg/ml after 48 hours of intervention;In this study,less than half of the inhibitory concentration was selected for formal experiments.The use concentration of Qingxuedu capsule was selected as three dose groups: low,medium and high(0.125,0.25 and 0.5 mg/ml)dose group.Compared with the control group,the apoptosis index of HaCaT cells increased significantly in Qingxuedu capsule medium dose group and Qingxuedu capsule high dose group(P<0.05,P<0.001),the protein expression of Shh、SMO and Gli1 decreased significantly in Qingxuedu capsule high dose group(P<0.05,P<0.01);the expression of Bcl-2 protein decreased significantly in Qingxuedu capsule low dose group,Qingxuedu capsule medium dose group and Qingxuedu capsule high dose group(P<0.05,P<0.01,P<0.001);the relative m RNA expressions of Bcl-2,Shh and Gli1 in Qingxuedu capsule high dose group decreased significantly(P<0.05,P<0.01),and the relative expression of SMOm RNA decreased significantly in Qingxuedu capsule low dose group,qingxuedu capsule medium dose group and Qingxuedu capsule high dose group(P<0.01,P< 0.001,P< 0.0001).3.Compared with the control group,the cell activity of rhshh induced group(0.5 μg/ml)was significantly increased(P<0.05),the apoptosis rate was significantly decreased(P<0.01),and the expression of Bcl-2protein and the relative expression of Bcl-2 m RNA were significantly increased(P<0.05,P<0.01);Compared with rhshh induction group(0.5μg/ml),the cell survival rate of rhshh induction(0.5 μg/ml)+Qingxuedu capsule group and rhshh induction(0.25 μg/ml)+Qingxuedu capsule group decreased significantly(P<0.01,P<0.05),and there was significant difference between rhshh induction(0.5 μg/ml)+Qingxuedu capsule group and rhshh induction(0.25 μg/ml)+Qingxuedu capsule group(P<0.01),the apoptosis rate of rhshh induced(0.25 μg/ml)+ Qingxuedu capsule group increased significantly(P<0.05),the protein expression of Bcl-2 in rhshh induced(0.25μg/ml)+Qingxuedu capsule group decreased significantly(P<0.01),and the relative expression of Bcl-2 m RNA in rhshh induced(0.25 μg/ml)+ Qingxuedu capsule group decreased significantly(P<0.01).[Conclusion]1.After treatment with Qingxuedu capsule,the symptoms and pathological changes of psoriasis mice were improved;Qingxuedu capsule has a significant effect on psoriasis,which is worthy of popularization and application.2.Qingxuedu capsule can inhibit HaCaT cell proliferation,promote HaCaT cell apoptosis;Qingxuedu capsule can reduce the protein expression of Shh,SMO,Gli1 and Bcl-2 in HaCaT cells and reduce the relative expression of Shh,SMO,Gli1 and Bcl-2 m RNA in HaCaT cells3.Rhshh negative regulation Qingxuedu capsule inhibits HaCaT cell proliferation and promotes HaCaT cell apoptosis.It is inferred that qingxuedu capsule plays a role in the treatment of psoriasis by inhibiting Shh signal pathway,down regulating the expression of Bcl-2,promoting HaCaT cell apoptosis and inhibiting HaCaT cell proliferation.