Study On The Function And Mechanisms Of Yin Xieling For Psoriasis By Regulating Langerhans And T Helper Cells

BackgroundPsoriasis is a common chronic inflammatory skin disease characterized by inflammatory infiltration,acanthosis of the epidermis,and hyperkeratosis.The pathogenesis of psoriasis is complex,and the exact mechanism is not completely clear yet.However,it is generally accepted that psoriasis is the result of genetic,epigenetic and environmental influences.Psoriatic lesions are characterized by excessive proliferation,incomplete differentiation of keratinocytes,obvious infiltration of inflammatory cells,proliferation of capillaries and so on.Recent studies have shown that psoriasis is mainly mediated by dendritic cells(DC)and T cells.Langerhans cells(LC),one of especial DC,are the specific antigen presenting cells in epidermal.It senses pathogen-related molecules through the connection pattern recognition receptor,produces a variety of cytokines,and presents antigens to T cells to stimulate specific T cell responses.Abnormal T cell activation due to dysregulation of DC is thought to be responsible for many disease-specific immune responses,such as psoriasis.Traditional Chinese medicine is considered that the treatment of psoriasis should be "from the perspective of blood and dryness".According to its characteristics,the experts summed up a lot of therapies with reliable efficacy and less adverse reactions.Ying Xie Ling(YXL)is an effective prescription summed up by Professor Guowei Xuan,based on his clinical experience.This formula was composed of Radix Paeoniae Rubra,Rhizoma smilacis glabrae,Herb of glabrous sarcandra,Rhizoma curcumae and Fructus mume.The combination of these herbs can nourish blood and moisten dryness,cool blood and detoxify,remove blood stasis and dredge collaterals Hence the prescription has achieved reliable clinical efficacy.Early research has shown that YXL can promote the formation of the epidermal granular layer,reduce the degree of itching and the area of skin wheals in mice,and inhibit macrophage polarization.However,there are few studies on the regulation of LC and T cells in psoriasis,so this research studied the immune regulation mechanism of YXL from the perspective of LC and T cells.ObjectiveTo explore the role and mechanism of YXL in regulating Langerhans cells and T helper cells.Furthermore,further research was conducted on the efficacy of Taxifolin,the main active component of YXL,in the treatment of psoriasis,and the regulatory effect and mechanism of T helper cellsMethods1.Establishment of psoriatic Balb/c mice model and observation on the therapeutic effect of YXL.Balb/c mice were randomLy divided into six groups,including Blank group,IMQ group,cyclosporine A group,and high dose,middle dose and low dose of YXL groups.mice were shaved on back,and then were oral treated for continuous 7 days.The high,medium and low dose of YXL groups were given 1.5g/kg,1g/kg,and 0.5g/kg Yinxieling,respectively.Cyclosporine A group was given 2mg/mL,and each group was administered at a dose of 0.2mL/10g.At the same time,the blank group and the IMQ group were given the same amount of water.From the third day,apply 50mg of 5%IMQ evenly on the back for 5 consecutive days,and record the body weight every day.The symptoms of erythema,scales,and thickening in the back of mice were also observed from the second day after modeling,and the PAS I scores were recorded calculated.On the eighth day,all the mice were sacrificed,skin leisions,skin draining lymph nodes were collected for the following determination.2.Study on the regulatory effect of YXL on LCs.Mice were given the drug by intragastric administration for 5 days and started modeling with IMQ on the forth day.After deep anesthesia on the sixth day,the cervical vertebra was dislocated and put to death.Skin and lymph nodes were taken in order to separate epidermal cells and lymphocytes.The expression of LC in epidermi cells and lymphocytes were detected and analyzed by flow cytometry.3.Study on the effects of YXL on T cells.Mice were oral administered for 7 days and started modeling with IMQ on the third day.On the eighth day,after deep anesthesia,the mice were sacrificed,and the skin lesions were used to observe the effects of YXL on the infiltration of CD3+T cells and CD4+T cells.Then RT-PCR was used to detect the relative expressions of the cytokines,IFN-γ,IL-4 and IL-17a,as well as their transcription factors,T-bet,GATA3 and ROR γt in skin lesions.Flow cytometry was used to detect the expression of T cell related cytokines and transcription factors in skin draining lymphocytes.4.Study on the effects and mechanisms of Taxifolin(TXL),the main active ingredient of YXL,on psoriasisHaCat cell line was employed to evaluate the anti-proliferation effect of TXL on LPS-induced abnormal proliferation.And then mice were given the TXL by gavage for 7 days.The dosage of TXL group was 40mg/kg and the CsA group was 2mg/mL,respectively.From the third day,50mg of 5%IMQ was used for modeling.On the eighth day,after deep anesthesia,mice were sacrificed.The thickness and the number of layers of epidermal,the degree of inflammatory cell infiltration was observed by skin pathological section.RT-PCR and flow cytometry were used to detect and analyze cytokines and transcription factors secreted by Th1/Th2/Th17 cells in skin lesions and lymph nodes.Proteins extracted from skin lesions were detected by Western Blot to investigate the mechanism of TXL in relieving the symptoms of psoriasis.Results1.After molding based on the above methods,the clinical symptoms such as erythema,scales and thickening were significantly aggravated,among which the PAS I scores were rised up to 11.00±0.82,the skin thickness was 275.54±14.64 μm,and the weight gain rate was-8.87%±1.63%.After administration of YXL,psoriasis-like lesions were significantly relieved.The PASI score in the medium-dose group was reduced to 6.25±1.71(P<0.05),the weight gain rate was-2.39%±1.72%(P<0.01),and the epidermal thickness was significantly reduced to 148.97±6.60 μm(P<0.001).These results suggest that YXL can effectively relieve the clinical symptoms and pathological changes of psoriasis.2.The results of flow cytometry showed that the expressions of PU.1 and LC were significantly decreased in YXL group(P<0.001),indicating that YXL could effectively inhibited the expression of LC and PU.1 in epidermis but did not affect the expression level of DC in lymph nodes(P>0.05).In addition,the ratio of LC migrated to lymph nodes was 2.17±2.47%in YXL group,while that in IMQ group was 40.60±7.66%,indicating that YXL could significantly inhibit the migration of LC from epidermis to lymph nodes(P<0.001).Meanwhile,the result also showed that YXL had inhibitory effect on antigen presentation of LC(P<0.05).Therefore,YXL may alleviate psoriasis by inhibiting the migration and the antigen presentation of LC.3.YXL reduced the infiltration of CD3+T cells and CD4+T cells in psoriatic lesions and the expressions of pro-inflammatory cytokines IL-17a and IFN γ(P<0.05),but promoted the secretion of IL-4,an Th2 type anti-inflammatory factor(P<0.05).The results of flow cytometry showed that the ratios of TCR β+ T cells and CD4+T cells were not significantly affected by YXL(P>0.05),but the percent of γ δ+T cells which could secrete IL-17a was significantly decreased(P<0.05).But further data showed that YXL had no significant effect on their transcription factors,RORγt and T-bet(P>0.05).On the contrary,YXL had an obvious promoting effect on Th2 cell cytokine IL-4 and its transcription factor,GATA3(P<0.01).Therefore,YXL may play a therapeutic role by promoting Th2 cell response and simultaneously inhibiting Th1 and Th17 cell responses.4.TXL could effectively inhibit the abnormal proliferation of HaCat cells induced by LPS(P<0.001)and alleviate the clinical and pathological symptoms of IMQ-induced psoriasis(P<0.05).TXL decreased the expression of pro-inflammatory factors IL-17a and IFN γ,and their transcription factors RORγt and T-bet.But significantly promoted the expression of anti-inflammatory cytokine IL-4 and its transcription factor GATA3(P<0.001).TXL also inhibited the expressions of Notch1,RBPJK,JAK2 and p-stat3(P<0.05).Therefore,TXL may promote the anti-inflammatory factor expression of IL-4 and its transcription factor expression of GATA3,while reduce the pro-inflammatory factors level of IL-17a and IFN γ as well as the transcription factors level of RORγt and T-bet by regulating Notchl pathway and JAK2/stat3 pathway.Conclusions1.YXL effectively alleviates the clinical and pathological symptoms of psoriasis,reduces its PASI score,and effectively rescues the weight.2.YXL reduces the expressions of LCs and PU.1 in psoriatic lesions and inhibits LC migration and its functions of antigen presentation.3.YXL dramatically reduces the infiltration of CD3+T cells and CD4+T cells in skin lesions.And promotes the differentiation of Th2 cells and inhibits those of Th1 and Th17 cells.4.TXL effectively inhibits the proliferation of HaCat cells and alleviats the clinical and pathological symptoms of psoriasis.By inhibiting the Notchl and JAK2/STAT3 signal pathways,it promots the expression of Th2 cells and inhibits the expressions of Th1 and Th17 cells.