Mechanism Of PSORI-CM02 Regulating The Proliferation Of Psoriatic Keratinocytes By MicroRNA-99a

Psoriasis is a common erythematous scaly skin disease characterized by an accelerated keratinocyte proliferation,parakeratosis,and a chronic inflammatory response to dermal lymphocyte infiltration.Among them,western medicine treatment of psoriasis has poor efficacy and toxic side effects,while Chinese medicine treatment of psoriasis is effective and has little side effects.Therefore,Chinese medicine treatment of psoriasis has certain advantages.Chinese medicine theory that Psoriasis is the main pathogenesis of hypothermia,blood heat,consumption of qi and blood,blood deficiency wind dry,blood heat mutual.Therefore,clinically cool Runzao,blood and blood nourishing agent are commonly used in the treatment of Psoriasis concept.PSORI-CM02 is from Professor Chuanjian Lu,in Guangdong Province Chinese medicine hospital Professor Guowei Xuan treatment of Psoriasis experience side Psoriasis based on the optimization of traditional Chinese medicine prescription,clinically used mainly for the treatment of blood stasis type Psoriasis.Our previous study has found that has-miR-99a changes significantly in the skin of patients with psoriasis,and the unbalanced expression of hsa-miR-99a may lead to abnormal proliferation and differentiation of psoriatic keratinocytes.thus,this study,starting from the overall concept,explores the molecular mechanism of PSORI-CM02 in the treatment of Psoriasis by in vivo and in vitro experiments.This study is divided into four parts:The first part,effect of PSORI-CM02 on Psoriasis-induced mice Induced by imiquimod(IMQ);The second part,mechanism of PSORI-CM02 on IMQ-induced Psoriasis model mice;the third part,effect of PSORI-CM02 on Interleukin-22/Interleukin-17A(IL-22/IL-17A)Mediating Psoriasis Model Cells;the fourth part,mechanism of PSORI-CM02 on IL-22/IL-17A Mediating Psoriasis Model Cells.Objective:To study the mechanism of PSORI-CM02 regulating the proliferation of psoriatic keratinocytes by microRNA-99a.Methods:Part one:Thirty Balb/c mice were divided into normal group,model group(IMQ,50 mg/only),PSORI-CM02 low dose group(1 g/kg),PSORI-CM02 High dose group(1.5 g/kg)and methotrexate(MTX,0.154 mg/kg)group.The rats in the model group and the administration group were dosed with IMQ continuously for 7 days on the back of the mice and the normal group were given the same amount of petrolatum three days after the administration of the medicine group(normal group and IMQ group were administered with the same volume of drinking water).PASI score was calculated on day 1,day 3,day 5,and day 7 of model establishment.Body weight and skin temperature were measured.The first 7 days of observation epidermal skin and spleen.The skin of the mice’s skin at the back was examined by RT-PCR for the detection of IL-23 mRNA,IL-8 mRNA,CCL20 mRNA and TNF-α mRNA expression.Part two:Thirty Balb/c mice were divided into normal group,model group(IMQ,50 mg/only),PSORI-CM02 low dose group(1 g/kg),PSORI-CM02 High dose group(1.5 g/kg)and methotrexate(MTX,0.154 mg/kg)group.The rats in the model group and the administration group were dosed with IMQ continuously for 7 days on the back of the mice and the normal group were given the same amount of petrolatum three days after the administration of the medicine group(normal group and IMQ group were administered with the same volume of drinking water).On day 7,the expression of mmu-miR-99a-5p(miRNA 99a)was detected by reverse transcription polymerase chain reaction(RT-PCR)in the skin of the back of the mice.The expressions of p-AKT,AKT,p-mTOR/mTOR and p-IKKα/IKK a,P-NF-κB p65/NF-κB p65 and p-Stat3/Stat3 respectively.The expression of mTOR was detected by immunohistochemistry.Part three:Human immortalized keratinocytes(HaCaT)were stimulated with IL-22 and IL-17A cytokines to induce the activation of Th17 cells in vitro.The HaCaT keratinocytes were divided into 5 groups:normal group,model group(IL-17A plus IL-22 stimulation),PSORI-CM02 low dose group(1 mg/mL),PSORI-CM02 high dose group(2 mg/mL),MTX group(40 μ M).The model group was stimulated with IL-17A(20 ng/mL)and IL-22(20 ng/mL)for 6h.PSORI-CM02 or MTX was added to the model group,together with IL-17A 6h after receiving the sample.The levels of IL-23 mRNA,IL-8 mRNA,CCL20 mRNA and TNF-α mRNA were detected by RT-PCR.Part four:HaCaT was divided into two groups,using miRNA 99a to overexpress or inhibit plasmids to act on keratinocytes under inflamed conditions in vitro,and observe the mechanism of action of PSORI-CM02.The first group(N=6)consisted of CON group,CON 99a inhibitor group,Model blank control group,Model 99a inhibitor group,PSORI-CM02 blank vector control group(2 mg/mL group),PSORI-CM02 miRNA 99a inhibitor vector group(2 mg/mL 99a inhibitor group).The second group(N=6)consisted of CON group,CON 99a mimic group,Model group blank control group,Model 99a mimic group,PSORI-CM02 blank vector control group(2 mg/mL group),PSORI-CM02 miRNA 99a mimic carrier group(2mg/mL 99a mimic group),blank control vector was mimic ncontrol.In the first group,the model group was stimulated with IL-17A(20 ng/mL)and IL-22(20 ng/mL)for 6 h after transfection with miRNA 99a inhibitor(100 nmol/L)or inhibitor ncontrol(100 nmol/L)The rats in the treatment group were injected with PSORI-CM02(2 mg/mL),IL-17A(20 ng/mL)and IL-22(20 ng/mL)In the second group,the model group was stimulated with IL-17A(20 ng/mL)and IL-22(20 ng/mL)for 6h,48h after transfection with miRNA 99a mimic(50 nmol/L)or mimic ncontrol(50 nmol/L).The drug group was injected with PSORI-CM02(2 mg/mL),IL-17A(20 ng/mL)and IL-22(20 ng/mL)The mRNA expression of miRNA 99a,IL-23,IL-8,CCL20,TNF-α and mTOR were detected by RT-PCR.The protein expression of p-AKT/AKT,p-mTOR/mTOR,p-IKKα/IKK α,p-NF-κB p65/p-Stat3/Stat3 were detected by Western blot.Results:Part one:Compared with the normal group,the mice in the untreated group showed signs of skin lesions such as dermatitis,erythema and scaly,body weight loss,spleen enlargement and skin temperature increase.The levels of IL-23 mRNA,IL-8 mRNA and CCL20 mRNA,TNF-α mRNA expression levels were significantly increased(P<0.05).Compared with model group,PSORI-CM02 group and MTX group lesion of skin such as infertility,erythema and scaly,as well as body weight loss,spleen enlargement and skin temperature increase;PSORI-CM02 group and MTX group IL-23 mRNA,IL-8 mRNA,CCL20 mRNA and TNFa mRNA expression were significantly down-regulated(P<0.05).Part two:Compared with the normal group,the skin thickening in the back of the model group was obvious and the expression of mTOR positive rate was significantly increased(P<0.05).The expressions of the ratio of p-AKT/AKT,p-mTOR/mTOR,p-IKKα/IKKα,p-NF-κB p65/NF-κB p65 and p-Stat3/Stat3 was significantly increased,and the expression of miRNA 99a was significantly decreased(P<0.05).Compared with the model group,the thickening of the back of the mice in the PSORI-CM02 group and the MTX group slowed down and the expression of mTOR positive rate was significantly decreased(P<0.05).The expressions of p-AKT/AKT,p-mTOR/mTOR,p-IKKα/IKKα,The proportion of NF-κB p65/NF-κB p65 and p-Stat3/Stat3 decreased significantly,and the expression of miRNA 99a increased significantly(P<0.05).Part three:Compared with normal group,the expression of IL-23 mRNA,IL-8 mRNA,CCL20 mRNA and TNF-α mRNA in model group increased significantly(P<0.05).Compared with the model group,the mRNA levels of IL-23 mRNA,IL-8 mRNA,CCL20 mRNA and TNF-α in PSORI-CM02 group were significantly decreased(P<0.05).Part four:Inhibition of miRNA 99a expression attenuated the effect of PSORI-CM02 on Psoriasis model cells,ie,the release of IL-23,IL-8,CCL20 and TNF-α in 2 mg/mL 99 a inhibitor group.The ratio of p-AKT/AKT,p-mTOR/mTOR,p-IKKα/IKK α,p-NF-κB p65/NF-κB p65 and p-Stat3/Stat3 increased significantly(P<0.05).Overexpression of miRNA 99a could make the model of Psoriasis model cells less obvious.Compared with Model group,the release of IL-23,IL-8,CCL20 and TNF-α in Model 99a mimic group was decreased,p-AKT/AKT,p-mTOR/mTOR,p-IKK α/IKK α,p-NF-κB p65/NF-κB p65 and p-Stat3/Stat3 decreased significantly(P<0.05).Conclusion:PSORI-CM02 inhibits the proliferation of psoriatic keratinocytes and inhibits the expression of effectors of Th17 cells such as IL-23,IL-8,CCL20 and TNF-α,indicating that PSORI-CM02 has a clear treatment for animal and cell models in this experiment.The improved effect may be related to the up-regulation of miR-99a inhibiting its target protein mTOR,inhibiting the mTOR/Stat3 pathway and inhibiting the activation of the Akt/IKK α/NF-κB p65 pathway.