Clinical Efficacy And Mechanism Of Wen’s Ershen Dihuang Decoction In The Treatment Of Psoriasis Vulgaris With Blood Dryness Syndrome

ObjectiveWe undertook this study to describe the clinical efficacy of Wen’s Ershen Dihuang Decoction of psoriasis with blood dryness syndrome,the mechanism of action may involve the EGFR/ERK/AQP3 pathway.MethodsClinical research:We randomly assigned psoriasis patients with blood dryness syndrome to experimental（received oral Wen’s Ershen Dihuang Decoction）and control groups（received oral Danggui Yinzi）.Jojoba oil was applied topically for all subjects.Treatments were continued for 12 weeks.Outcome measures comprised Psoriasis Area and Severity Index（PASI）score,the Total effective rate,Body surface area（BSA）,Physician’s global assessment（PGA）score,pruritus visual analogue scale（VAS）score,TCM syndrome scores,Psoriasis main symptoms scale score and Psoriasis quality of life scale（PQOLS）score.The intelligent skin tester was used to measure the moisture and oil content of target skin lesions.Safety was also assessed.Peripheral blood samples of subjects were collected to measure aquaporin（AQP）3 m RNA by Real-time PCR.Animal experiment:54 BALB/c mice were randomly divided into 9 groups,A group（blank group）,B group（model group）,C group（Wen’s Ershen Dihuang Decoction group）,D group（NSC228155 group）,E group（Wen’s Ershen Dihuang Decoction+NSC228155 group）,F group（Bortezomib group）,G group（Wen’s Ershen Dihuang Decoction+Bortezomib）,H group（PD153035 group）,I group（U0126 group）.After shaving the back area of mice,all the groups were smeared with 62.5 mg of 5%imiquimod cream for 8 consecutive days,except for group A.Groups D（NSC228155group）and E（Wen’s Ershen Dihuang Decoction+NSC228155 group）were intraperitoneally injected with EGFR agonist NSC228155,groups F（Bortezomib group）and G（Wen’s Ershen Dihuang Decoction+Bortezomib）were injected intraperitoneally with the ERK agonist Bortezomib,1 mg·kg^-1·d^-1,twice a week.Group H（PD153035group）was injected intraperitoneally with EGFR inhibitor PD153035,8 mg·kg^-1·d^-1.Group I（U0126 group）was intraperitoneally injected with ERK inhibitor U0126,3mg·kg^-1·d^-1;Group C（Wen’s Ershen Dihuang Decoction group）,E（Wen’s Ershen Dihuang Decoction+NSC228155 group）,G（Wen’s Ershen Dihuang Decoction+Bortezomib）were intragastrically administered Wen’s Ershen Dihuang Decoction,38.64g·kg^-1·d^-1.Other mice were gavaged with UP water,0.4 ml·d^-1 for 8 consecutive days.On the last day,the mice were evaluated and scored using PASI,the water and oil content were measured by the intelligent skin tester.The weight of the kidney,lung,spleen and body were recorded to calculate the organ index.The pathologic changes in skin tissue were observed by hematoxylin-eosin（HE）stain and Baker score.The expression level of AQP3 m RNA in peripheral blood of mice in groups A（blank group）,B（model group）,C（Wen’s Ershen Dihuang Decoction group）were measured by real-time PCR.Immunohistochemical staining was used to detect the expression of AQP3in kidneys,lungs,and skin lesions in groups A（blank group）,B（model group）,C（Wen’s Ershen Dihuang Decoction group）.The expression level of AQP3,EGFR,p-EGFR,ERK and p-ERK in skin tissue were detected by Western Blot.ResultsClinical research:（1）PASI score:At the week 12,two groups had a reduction from baseline in the PASI score（P<0.01）.The experimental group had a greater reduction in the PASI score,as compared to control group（P<0.01）.（2）Clinical efficacy:At the week 12,the total effective rate of the experimental group was higher than that of the control group（P<0.05）.（3）BSA:The BSA of the two groups of subjects showed decreased compared with baseline（P<0.01）;The decrease of the BSA in the experimental group was better than in the control group（P<0.01）.（4）PGA score:Compared with baseline,the experimental group and control group attenuated the PGA score（P<0.01）.The amelioration of PGA score in the experimental group was better than that in the control group（P<0.05）.（5）Pruritus VAS:Compared with pre-treatment,the pruritus VAS of the subjects in the two groups was significantly decreased（P<0.01）.The pruritus VAS score of the experimental group was better than the control group at the week 12 point（P<0.05）.（6）TCM syndrome scores:The data indicated that the TCM syndrome scores of the two groups decreased（P<0.01）after treatment.The improvement of TCM syndrome scores in the experimental group was better than the control group（P<0.05）.（7）Psoriasis main symptom scale scores:The main symptom scale scores of the two groups decreased compared to the baseline（P<0.01）.The psoriasis main symptom scale score of the experimental group was lower than the control group（P<0.05）.（8）PQOLS score:The PQOLS scores of the two groups were decreased compared to pre-treatment（P<0.01）.The PQOLS score of the experimental group decreased slightly than the control group,which was not statistically significant.（9）AQP3 m RNA expression:Compared with baseline,the peripheral blood AQP3m RNA levels of the subjects in the control group and experimental group（P<0.01）were increased.Compared to the control group,the experimental group increased the level of AQP3 m RNA in peripheral blood of psoriasis patients with blood dryness syndrome（P<0.05）.The results showed a correlation in AQP3 m RNA expression and PASI score,r_s=-0.649,P=0.002.（10）Water content:At week 12,the control group and experimental group elevated the water content of skin lesions from baseline（P<0.01）;The experimental group significantly increased the water content than the control group at the endpoint（P<0.01）.（11）Oil content:In comparison with pre-treatment,the oil content of skin lesions in both groups was increased after treatment（P<0.01）;Results displayed the oil content of the experimental group was higher than the control group（P<0.05）.Animal experiment:（1）PASI score:Group A（blank group）had no skin lesions,and the PASI score was 0.Compared with group A,group B（model group）（P<0.05）,group D（NSC228155 group）（P<0.01）,group E（Wen’s Ershen Dihuang Decoction+NSC228155 group）（P<0.05）,group F（Bortezomib group）（P<0.01）,group G（Wen’s Ershen Dihuang Decoction+Bortezomib）（P<0.05）,and group H（PD153035 group）（P<0.05）increased PASI score.Compared with group B（model group）（P<0.05）,group D（NSC228155 group）（P<0.01）,and group F（Bortezomib group）（P<0.01）,group C（Wen’s Ershen Dihuang Decoction group）effectively reduced the PASI score of psoriasis animal model.Compared with group D（group NSC228155）,group H（group PD153035）had a lower PASI score（P<0.01）;group G（Wen’s Ershen Dihuang Decoction+Bortezomib group）had a lower PASI score compared with group F（Bortezomib group）（P<0.01）.（2）Histopathological features and Baker score:The epidermal structure of mice in group A（blank group）was complete.Group B（model group）,group D（NSC228155group）,and group F（Bortezomib group）showed obvious spines in the epidermis of layer hypertrophy,the number of layers increased,the epidermis elongated in a clubbing shape,hyperkeratosis,parakeratosis,telangiectasia,hyperemia and lymphocyte infiltration were seen in the dermis.Combined with the above PASI scores,the model was successfully replicated.The histopathological changes in group C（Wen’s Ershen Dihuang Decoction）were significantly relieved.Baker score:The Baker score of group A（blank group）was 0.Compared with group A（blank group）,group B（model group）（P<0.01）,group C（Wenshi Ershen Dihuang Decoction group）（P<0.01）,group D（NSC228155 group）（P<0.01）,group E（Wenshi Ershen Dihuang Decoction+NSC228155 group）（P<0.01）,group F（Bortezomib group）（P<0.01）,group G（Wenshi Ershen Dihuang Decoction+Bortezomib）（P<0.05）,group H（PD153035 group）（P<0.01）,and group I（U0126group）（P<0.01）elevated the Baker score.Compared with group B（model group）（P<0.05）,group D（NSC228155 group）（P<0.01）,and group F（Bortezomib group）（P<0.01）,group C（Wen’s Ershen Dihuang Decoction）significantly reduced the Baker score of animal skin lesions.Compared with group D（NSC228155 group）,group H（PD153035 group）（P<0.05）had a lower Baker score;compared with group F（Bortezomib group）,group I（U0126 group）（P<0.01）had a lower Baker score.（3）Organ index:The results showed no difference in the lung organ index and kidney organ index among the groups.Compared with group A（blank group）,group B（model group）（P<0.01）,group D（NSC228155 group）（P<0.01）,group E（Wen’s Ershen Dihuang decoction+NSC228155 group）（P<0.05）,group F（Bortezomib group）（P<0.01）,group G（Wen’s Ershen Dihuang decoction+Bortezomib）（P<0.01）,group H（PD153035 group）（P<0.05）,group I（U0126 group）（P<0.01）increased the spleen organ index.Group C（Wen’s Ershen Dihuang Decoction）could down-regulate the spleen organ index.（4）AQP3 m RNA expression in peripheral blood:Compared with group A（blank group）,group B（model group）（P<0.05）and group C（Wen’s Ershen Dihuang Decoction group）decreased the expression level of AQP3 m RNA.Compared with group B（model group）,group C（Wen’s Ershen Dihuang Decoction group）（P<0.05）had higher level of AQP3 m RNA in peripheral blood,which was consistent with the trend of clinical trial results.（5）Water and oil content of skin lesions:Compared with group A（blank group）,group B（model group）（P<0.01）,group D（NSC228155 group）（P<0.01）,group F（bortezomib group）（P<0.01）,group G（Wen’s Ershen Dihuang Decoction+Bortezomib）（P<0.05）,group H（PD153035 group）（P<0.05）,and group I（U0126 group）（P<0.05）decreased the water content of skin lesions.Compared with group B（model group）（P<0.05）,group D（NSC228155 group）（P<0.05）,group F（Bortezomib group）（P<0.05）,and group C（Wen’s Ershen Dihuang Decoction group）increased water content of skin lesion.Compared with group A（blank group）,group B（model group）（P<0.01）,group D（NSC228155 group）（P<0.01）,group F（Bortezomib group）（P<0.05）and other groups had lower oil content in skin lesions;Compared with group B（model group）,group C（Wen’s Ershen Dihuang Decoction group）increased the oil content of skin lesions（P>0.05）.（6）AQP3（IOD）expression in kidney,lung and skin:Compared with group A（blank group）,group B（model group）（P<0.01）and group C（Wen’s Ershen Dihuang Decoction group）（P<0.05）had a statistically significant decrease in the expression of AQP3（IOD）expression in kidneys;Compared with group B,group C（P<0.05）increased AQP3（IOD）expression.Compared with group A（blank group）,group B（model group）（P<0.01）and group C（Wen’s Ershen Dihuang Decoction group）（P<0.05）decreased the expression of AQP3（IOD）in the lungs;Group C（Wen’s Ershen Dihuang Decoction group）（P<0.05）increased the expression of AQP3（IOD）.Compared with group A（blank group）,group B（model group）（P<0.01）,group C（Wen’s Ershen Dihuang Decoction group）（P>0.05）down-regulated the AQP3（IOD）expression;compared with group B,group C（Wen’s Ershen Dihuang Decoction group）（P<0.05）increased AQP3（IOD）expression in the skin.（7）EGFR,p-EGFR,ERK,p-ERK,AQP3 protein expression in skin tissue:Compared with group A（blank group）,group B（model group）,group D（NSC228155 group）,and group F（Bortezomib group）decreased the expression of AQP3;Compared with group B（model group）（P<0.05）,group D（NSC228155 group）（P<0.01）and group F（P<0.05）,group C（Wen’s Ershen Dihuang Decoction group）significantly increased the expression of AQP3.Compared with group A（blank group）,group B（model group）（P<0.01）and group D（NSC228155 group）（P<0.01）increased the expression level of total EGFR in skin tissue.The level of total EGFR in group D（NSC228155 group）（P<0.05）was higher than in group B（model group）;Compared with group B（model group）,group C（Wen’s Ershen Dihuang Decoction group）（P<0.01）decreased the expression level of EGFR protein.Compared with group D（NSC228155 group）,group E（Wen’s Ershen Dihuang Decoction+NSC228155 group）（P<0.01）and group H（PD153035 group）（P<0.01）decreased the expression level of EGFR protein.The expression of p-EGFR in group B（model group）（P<0.01）,and group D（NSC228155 group）（P<0.01）were higher than in group A（blank group）.Compared with group B（model group）,group C（Wen’s Ershen Dihuang Decoction group）（P<0.05）decreased the level of p-EGFR.Compared with group D（NSC228155 group）,the expression of p-EGFR was down-regulated in group E（Wen’s Ershen Dihuang Decoction+NSC228155 group）.The expression of p-EGFR in group D（NSC228155group）was higher than in group H（PD153035 group）（P<0.05）.The expression of ERK in groups B（model group）and F（Bortezomib group）was enhanced than in group A（blank group）;Compared with group B（model group）,group C（Wen’s Ershen Dihuang Decoction group）down-regulated the expression of ERK in skin lesions;Group G（Wen’s Ershen Dihuang Decoction+Bortezomib）down-regulated the level of ERK elevated by agonists.Compared with group A（blank group）,the level of p-ERK increased in group B（model group）,group D（group NSC228155）,and group F（group Bortezomib）.While group C（Wen’s Ershen Dihuang Decoction group）,group E（Wen’s Ershen Dihuang Decoction+NSC228155 group）,group G（Wen’s Ershen Dihuang decoction+Bortezomib）,group H（PD153035 group）,and group I（U0126 group）down-regulated the level of p-ERK.ConclusionClinical research:（1）Wen’s Ershen Dihuang Decoction can safely and effectively reduce the PASI score of skin lesions in psoriasis patients with blood dryness syndrome and improve the total clinical rate,decrease the BSA,PGA score,relieve itching,TCM syndrome scores and the main symptoms of psoriasis Scale,reduce the PQOLS score.（2）Wen’s Ershen Dihuang Decoction increased the expression of AQP3 m RNA in peripheral blood,water and oil content of skin lesions.To sum up,under the guidance of the theory of"skin and hair generate kidney",Wen’s Ershen Dihuang Decoction can safely and effectively improve the clinical manifestations of psoriasis vulgaris and ameliorate the dryness of the skin lesions.Animal experiments:（1）Wen’s Ershen Dihuang Decoction could effectively improve the PASI score,histopathological features in model mice,reduce Baker score,do no obvious damage to lung,kidney and spleen.（2）Wen’s Ershen Dihuang Decoction increased the AQP3 m RNA expression in peripheral blood,water,and oil content of skin lesion,simultaneously increased the expression levels of AQP3 in the lungs and kidneys.（3）Wen’s Ershen Dihuang Decoction could down-regulate the levels of EGFR,ERK,p-EGFR,and p-ERK in psoriatic skin lesions,and up-regulate the expression of AQP3.Combined with clinical trials,the therapeutic effect of Wen’s Ershen Dihuang Decoction on psoriasis vulgaris may involve the EGFR/ERK/AQP3 pathway.