Study On The Clinical Efficacy And Mechanism Of Wen’s Qingxin Kaixuan Formula In The Treatment Of Psoriasis Vulgaris With Blood-Heat Syndrome Based On The Theory Of "Heart Commanding The Exterior"

Objective: This study aims to observe the clinical efficacy of Wen’s Qingxin Kaixuan formula in the treatment of psoriasis vulgaris with the blood-heat syndrome under the guidance of the theory of “heart commanding the exterior”,so as to enrich the theoretical connotation and clinical application of “heart commanding the exterior”,and promote the experience of Wen’s dermatological school in Sichuan in the treatment of psoriasis.The study also aims to discuss the possible mechanism of Wen’s Qingxin Kaixuan formula in the treatment of psoriasis through mechanism research,and then explain the scientific connotation of the theory of “heart commanding the exterior”.Methods:1.Part one: Taking the randomized controlled trial as the research design,76 psoriasis vulgaris subjects with the blood-heat syndrome who met the inclusion criteria were randomly divided into the treatment group(Wen’s Qingxin Kaixuan formula combined with urea ointment)and control group(Xijiao Dihuang decoction combined with urea ointment).There were 38 cases in each group and the course of treatment was 8 weeks.Psoriasis Area and Severity Index(PASI),Physician’s Global Assessment(PGA),Body Surface Area(BSA),and Visual Analogue Scale(VAS)were scored at the baseline period,the 4th week,and 8th week after enrollment.Dermatology Life Quality Index(DLQI),Psoriasis Disability Index(PDI),and Psoriasis Vulgaris Main Symptom Scale were scored at the baseline period and 8th week after enrollment.The safety of Wen’s Qingxin Kaixuan formula was evaluated by adverse events during treatment.2.Part two: 10 subjects in the treatment group and 10 subjects in the control group were randomly selected to obtain the anticoagulant whole blood samples before and after treatment.q PCR was used to detect the m RNA expression of RORγt and Foxp3 in the peripheral blood of these subjects.Pearson and Spearman correlation coefficients were used to analyze the correlation between the m RNA expression of RORγt and Foxp3,the ratio of RORγt/Foxp3,and the PASI score before treatment.3.Part three: Using imiquimod cream-induced psoriasis-like mice as the animal model,30 BALB/c male mice were randomly divided into blank group(Control),model group(Model),Hippo pathway inhibitor group [XMU-MP-1(1mg/kg)],Qingxin Kaixuan formula group [QXKX(24.25g/kg)] and Hippo pathway inhibitor + Qingxin Kaixuan formula group [XMU-MP-1(1mg/kg)+ QXKX(24.25g/kg)].Each group has six mice,which were given oral gavage once a day for 7 days.Mice in Control and Model groups were given an equivalent volume of UP water.The bodyweight of mice was measured every day,and the back skin condition was also observed.The PASI score of the mice’s skin lesions was scored at the end of the experiment on the 7th day.Mice were sacrificed by anesthesia,the spleen tissues were taken and weighed,the spleen index of the mice in each group was calculated,and the proportion of Th17 and Treg cells in the spleen was detected by flow cytometry;the back skin tissues were collected,and the skin pathological changes of the mice in each group were observed by HE staining.Meanwhile,the Baker scores were calculated.The m RNA expressions of RORγt and Foxp3 in the skin tissues were detected by the q PCR method,and the protein expressions of Hippo pathway molecules such as MST1/2,YAP,and TAZ in the skin tissues were detected by Western Blot.Results:1.Part one:(1)Comparison of study completion and baseline data: A total of 76 PV subjects with the blood-heat syndrome were recruited,of which 5 cases fell off(1 case in the treatment group and 4 cases in the control group),and 71 cases were completed.There was no significant difference in the completion between the two groups(P>0.05).The demographic data,disease characteristics,and other baseline data of the two groups were comparable(P > 0.05).(2)Evaluation of the skin lesion severity: the response rate of PASI 50 in the treatment group was significantly higher than that in the control group(P<0.05),and the response rate of PASI 75 in the treatment group was higher than that in the control group,but the difference was not statistically significant(P>0.05).The results of the generalized estimation model(GEE)of PASI score showed that the overall PASI scores of different interventions and visit times were significantly different(P<0.05),and the significant difference in the speed of reducing PASI scores between the two groups was also found(P<0.05).Further comparison between groups showed that the PASI scores of the treatment group at the 4th and 8th week after enrollment were significantly lower than those of the control group(P<0.05),and the most obvious difference in PASI score between the two groups was found at the 8th week after enrollment.The GEE results of the PGA score showed that the overall PGA scores of different interventions and visit times were significantly different(P<0.05),and a significant difference in the speed of reducing the PGA score between the two groups was also found(P<0.05).Further comparison between groups showed that the PGA score of the treatment group was significantly lower than that of the control group at the 8th week after enrollment(P<0.05),while there was no significant difference between the two groups at the 4th week after enrollment(P>0.05),and the significant difference of PGA score between the two groups began to appear at the 8th week after enrollment.The GEE results of the BSA score showed that the overall BSA scores of different visit times were significantly different(P<0.05),while the effects on the BSA score of intervention and the interaction between intervention and visit time were not statistically significant(P>0.05).Further comparison between groups showed that the BSA score of the treatment group was significantly lower than that of the control group at the 8th week after enrollment(P<0.05),while there was no significant difference between the two groups at the 4th week after enrollment(P>0.05),and the significant difference of BSA score between the two groups began to appear at the 8th week after enrollment.(3)Evaluation of the clinical symptom: the GEE results of the VAS pruritus score showed that the overall VAS pruritus scores of different interventions and visit times were significantly different(P<0.05),and the significant difference in the speed of reducing VAS pruritus score between the two groups was also found(P<0.05).Further comparison between groups showed that the VAS pruritus scores of the treatment group at the 4th and 8th week after enrollment were significantly lower than those of the control group(P<0.05),and the most obvious difference in VAS pruritus scores between the two groups was found at the 8th week after enrollment.The scores of the PV main symptom scale in the treatment group and control group after treatment were significantly lower than that before treatment(P<0.05).The comparison results between the two groups showed that the score of the PV main symptom scale in the treatment was significantly lower than that in the control group(P<0.05).(4)Evaluation of the quality of life: the DLQI and PDI scale scores of the treatment group and the control group after treatment were significantly lower than those before treatment(P<0.05).The results of the comparison between groups showed that the DLQI and PDI scale scores of the treatment group after treatment were lower than those of the control group,and the difference in DLQI scale scores between the two groups was statistically significant(P<0.05),while the difference of PDI scale score was not statistically significant(P>0.05).(5)Evaluation of the safety: there was no significant difference in the incidence of adverse events between the treatment group and the control group(P>0.05).2.Part two:(1)The m RNA expression of RORγt and RORγt/Foxp3 ratio in peripheral blood of subjects with psoriasis vulgaris before treatment were positively correlated with PASI score(P<0.05),while the m RNA expression of Foxp3 in peripheral blood before treatment was negatively correlated with PASI score(P<0.05).(2)The PASI score and the m RNA expression of RORγt in peripheral blood after treatment in the treatment group and control group were significantly lower than those before treatment(P<0.05),while the m RNA expression of Foxp3 in peripheral blood after treatment was significantly higher than those before treatment(P<0.05).The comparison between the two groups showed that the PASI score and the m RNA expression of RORγt in peripheral blood after treatment in the treatment group were significantly lower than those in the control group(P<0.05),while the m RNA expression of Foxp3 in peripheral blood after treatment was significantly higher than that in the control group(P<0.05).3.Part three:(1)Comparison of the skin lesions: compared with the Control group,the exposed skin on the back of mice in the Model group and XMU-MP-1 group showed obvious thickening of skin lesions with large areas of erythema and scales,which were similar to human psoriasislike skin lesions.The total PASI score of skin lesions in the two groups was significantly increased(P < 0.05).Compared with the Model group and XMU-MP-1 group,the symptoms of skin lesions in the QXKX group were significantly improved,and the total PASI score was significantly reduced(P < 0.05);The skin lesions in the XMU-MP-1+ QXKX group had a certain degree of improvement,and the total PASI score decreased,but the difference was not statistically significant(P > 0.05).The total PASI score of mice in the QXKX group was lower than that in the XMU-MP-1+QXKX group,but the difference was not statistically significant(P>0.05).(2)Comparison of the body weight and spleen index: compared with the Control group,the mice weight in the other groups was significantly reduced,the spleen was significantly enlarged,and the spleen index was significantly increased(P < 0.05).The mice weight in the Model group,XMU-MP-1 group,XMU-MP-1+ QXKX group showed the trend of XMU-MP-1+ QXKX > Model group > XMU-MP-1 group.The weight differences among these three groups were not statistically significant(P > 0.05).The spleen index of these three groups showed the trend of XMU-MP-1+QXKX group <Model group <XMU-MP-1 group,only the difference in spleen index between XMU-MP-1 group and XMU-MP-1+QXKX group was statistically significant(P<0.05).Compared with these three groups,the mice weight in the QXKX group was significantly decreased(P<0.05),while the spleen index was increased,and only the difference in the mice weight between the QXKX group and XMU-MP-1+QXKX group was not statistically significant(P>0.05).(3)Comparison of the pathological change of skin lesions and Baker score: compared with the Control group,the skin lesions of the Model group and XMU-MP-1 group showed obvious psoriasis-like pathological histomorphology,and the Baker scores were significantly increased(P < 0.05).Compared with the Model group and XMU-MP-1 group,the psoriasislike pathological histomorphology of mice in the XMU-MP-1+QXKX group was improved,and the Baker score was significantly reduced(P<0.05).In the QXKX group,the improvement of psoriasis-like pathological histomorphology was the most obvious,and the Baker score was significantly decreased(P < 0.05).Compared with the QXKX group,the psoriasis-like pathological histomorphology of mice in the XMU-MP-1+QXKX group was more obvious,and the baker score was significantly higher(P < 0.05).(4)Comparison of the outcomes of Th17/Treg balance: compared with the Control group,the proportion of Th17 cells in the spleen and the m RNA expression of RORγt in the skin lesions in the Model group,XMU-MP-1 group,and XMU-MP-1+QXKX group were significantly increased,while the proportion of Treg cells in the spleen and the m RNA expression of Foxp3 in the skin lesions were significantly decreased(P<0.05).The proportion of Th17 cells in the spleen and the m RNA expression of RORγt in the skin lesions of the three groups showed the trend of XMU-MP-1 group > Model group > XMU-MP-1 + QXKX group,while the proportion of spleen Treg cells and the m RNA expression of Foxp3 in skin lesions showed the trend of XMU-MP-1 group < Model group < XMU-MP-1+QXKX group.There were no significant differences in the above outcomes among these three groups(P>0.05).Compared with these three groups,the proportion of Th17 cells in the spleen and the m RNA expression of RORγt in the skin lesions in the QXKX group were significantly decreased,while the proportion of Treg cells in the spleen and the m RNA expression of Foxp3 in the skin lesions were significantly increased(P<0.05).(5)Comparison of the protein expression of Hippo pathway molecules: compared with the Control group,the protein expression of MST1/2 in the skin lesions of the Model group and XMU-MP-1 group was significantly decreased,while the protein expression of YAP and TAZ was significantly increased(P<0.05).The protein expression of MST1/2 in the Model group,XMU-MP-1 group,and XMU-MP-1+QXKX group showed the trend of XMU-MP-1+QXKX group≈Model group>XMU-MP-1 group,while the protein expression of YAP and TAZ showed the trend of XMU-MP-1+ QXKX group<Model group<XMU-MP-1 group.There was no significant difference in the above outcomes among these three groups(P>0.05).Compared with these three groups,the protein expression of MST1/2 in the QXKX group was significantly increased,and the protein expression of YAP was significantly decreased(P<0.05).The protein expression of TAZ in the QXKX group was decreased,however,there was no significant difference between the QXKX group and XMU-MP-1+QXKX group(P>0.05).Conclusion:1.Wen’s Qingxin Kaixuan formula can significantly improve the skin lesion severity,clinical symptoms,and quality of life of patients who were diagnosed as PV with blood heat syndrome,and has good clinical efficacy and safety.2.Wen’s Qingxin Kaixuan formula based on the theory of "heart commanding the exterior" has therapeutic advantages in the treatment of PV with blood heat syndrome,the response rate of PASI 50 and improvement of the PASI score,PGA score,clinical symptoms,DLQI scale is better than that of Xijiao Dihuang Decoction.3.The imbalance of expression ratio between RORγt and Foxp3 can reflect the severity of patients who are diagnosed as PV with the blood-heat syndrome to a certain extent.Wen’s Qingxin Kaixuan formula may play a therapeutic role by regulating the abnormal expression of RORγt and Foxp3,thereby regulating the immune balance of Th17/Treg in the body.4.Wen’s Qingxin Kaixuan formula can significantly improve the appearance and pathological histomorphology of psoriasis-like mice,reduce the total PASI score,Baker score,and spleen index,and restore the immune function of mice.5.Wen’s Qingxin Kaixuan formula may play a role in regulating the immune imbalance of Th17/Treg in psoriasis-like mice by activating the Hippo pathway.6.The inhibition of Hippo pathway activity may be related to the pathogenesis of psoriasis.Its role in regulating the immune balance of Th17/Treg in psoriasis has not been clear,which needs to be confirmed by further research.