| Objective:This study was performed to clarify the mechanism of action of JWLX on its source mi R-203a-3p,secondary inhibitory factor SOCS3,and downstream mitochondrial apoptosis pathway through two levels,human and animal,with JAK2/STAT3 pathway as the core,to provide a shred of more comprehensive biological evidence for its clinical application in the treatment of blood-heat syndrome psoriasis and to enrich the dialectical treatment of psoriasis in Chinese medicine material basis.Methods:1.Clinical trial: 13 patients with blood-heat psoriasis(treatment group)and 8healthy subjects(control group)were recruited through an open-label trial.JWLX was administered to the treatment group for 8 weeks without intervention in the control group.mi R-203-3p,SOCS3,JAK2,and STAT3 m RNA expressions were detected in skin lesions before and after treatment in the treatment group and the control group by RT-q PCR.STAT3 m RNA expression,and observe the changes in PASI score、DLQI score,and safety index before and after treatment in the treatment group.And correlation analysis was performed to explore the correlation between SOCS3 and mi R-203-3p,STAT3 m RNA in skin lesions of patients with blood-heat evidence of psoriasis.Detailed documentation of the occurrence of adverse events(incisional healing and other treatment-related adverse events).2.Animal experiment: 36 BALB/c male mice were randomly divided into the blank group(Blank group),model group(Model group),JWLX high-dose group(JWLX-H group),medium-dose group(JWLX-M group),low-dose group(JWLX-L group),and methotrexate group(MTX group),and the hair on the back of all mice was removed from an area of about 2cm×3cm,except All the mice in each group except the Blank group were coated with 5% imiquimod cream(IMQ)for 7 days,and the intervention group was given the corresponding drug intervention at the same time of modeling.The effect of JWLX on the PASI score and Baker score of each group was observed by photographing and recording the skin lesions of mice and HE staining;the expression of epidermal proliferation index Ki-67 in mice was detected by immunohistochemistry and the apoptosis of epidermal keratinocytes in mice was detected by TUNEL.The expression of Bcl-2,Bax,Cyt-c,and Caspase-3 m RNAs in the mi R-203-3p-SOCS3 signaling axis and mitochondrial apoptosis pathway in mouse skin lesions were detected by RT-q PCR,and the expression of JAK2/p-STAT3 pathway and mitochondrial apoptosis Bcl-2,Bax,Cyt-c,Caspase-3 m RNAs in mouse skin lesions were detected by Western blot.Mitochondrial apoptotic Bcl-2,Bax,Cyt-c,and Cleaved caspase-3 protein expression were detected by Western blot.Results:1.Clinical trials: The PASI scores and DLQI scores of patients in the treatment group were significantly lower after treatment than before treatment(all p < 0.001);compared with the control group,the levels of mi R-203a-3p,JAK2,and STAT3 m RNA in the skin lesions before treatment were significantly higher in the treatment group,and a statistically significant difference was observed(all p < 0.001);meanwhile,the expression of SOCS3 m RNA was lower than showed a statistically significant difference(p < 0.001).After treatment with JWLX in the treatment group,JAK2 with STAT3 m RNA relative expression decreased significantly compared with that before treatment,the difference was significant(p<0.05,p<0.001),and SOCS3 m RNA expression increased compared with that before treatment,the difference was statistically significant(p<0.001);for mi R-203a-3p,the relative m RNA expression after treatment decreased,however,there was no statistically significant difference(p>0.05).The relationships between SOCS3 and mi R-203a-3p and STAT3 m RNA were all negatively correlated.All subjects had a satisfactory incisional recovery.Patients in the treatment group had no significant abnormalities in safety indicators before and after treatment,and no serious adverse events occurred.2.Animal experiments:(1)The effect of JWLX on PASI score and Baker score in psoriasis-like mice: PASI score and Baker score in each dose group of JWLX and MTX group decreased compared with Model group,and differences showed statistical significance(all p<0.001).Among them,no statistically significant difference was found between the JWLX-H group and JWLX-M group compared with the PASI score of the MTX group(all p>0.05),and no statistically significant difference was found between the JWLX-H group compared with Baker score of MTX group(p>0.01).(2)The effect of JWLX on Ki-67 positive rate and AI index in psoriasis-like mice:the epidermal Ki-67 positive cell rate was significantly higher in the Model group compared with the Blank group(p < 0.01);the Ki-67 positive cell rate was lower in all intervention groups compared with the Model group,and differences were statistically significant(p < 0.001),among which the JWLX-H group,the JWLX-M group,and MTX group showed no statistically significant difference between them(all p > 0.05).In response to apoptosis,the AI index of mice in the Model group was significantly higher than that in the Blank group(p < 0.05),and the AI index of mice in the MTX,JWLX-H,and JWLX-M groups was significantly higher than that in the Model group after intervention(all p < 0.01).The PASI score of mice was positively correlated with Ki-67 positivity and negative correlation with AI index.(3)Regulation of mi R-203a-3p-SOCS3-JAK2/STAT3 pathway in psoriasis-like mouse skin lesions by JWLX:The expression of mi R-203a-3p m RNA and JAK2 and p-STAT3 proteins were significantly higher in skin lesions of mice in the Model group compared with the Blank group(all p < 0.001);the expression of mi R-203a-3p m RNA and JAK2 proteins were significantly lower in the JWLX-H,JWLX-M,and MTX groups compared with the Model group(all p < 0.05).For p-STAT3,the p-STAT3 protein expression was significantly decreased in each JWLX group and MTX group compared with the Model group(all p < 0.05).For SOCS3,SOCS3 m RNA expression in skin lesions was significantly decreased in the Model group compared with the Blank group(p < 0.001),and SOCS3 m RNA expression was significantly increased in each JWLX group and MTX group compared with the Model group(both p < 0.05).(4)The regulation of JWLX on the expression of Bcl-2,Bax,Cyt-c,Caspase-3and its activated form Cleaved caspase-3 in the mitochondrial apoptotic pathway in psoriasis-like mouse lesions: the relative expression of genes and proteins of anti-apoptotic Bcl-2 in mouse lesions was significantly higher in the Model group compared to the Blank group(p < 0.001)and in the Compared with the Model group,the JWLX-H,JWLX-M,and MTX groups all significantly downregulated their gene levels and protein contents,and the differences were statistically significant(all p <0.05),among which the differences between the JWLX-H and MTX groups were not statistically significant(p > 0.05).The expression of pro-apoptotic factors Bax,Cyt-c,Caspase-3,and Cleaved caspase-3 in the skin lesions of mice in the Model group was significantly lower compared with that in the Blank group(all p < 0.001).For Bax,the gene levels and protein expression were upregulated in all JWLX groups and MTX groups compared with the Model group,and the differences were statistically significant(all p < 0.05).Cyt-c,JWLX-H,JWLX-M groups,and MTX groups could significantly up-regulate its gene level and protein expression,and the differences were statistically significant(all p < 0.001).For Caspase-3,only the JWLX-H group could upregulate its gene level in the herbal intervention group,and the difference was statistically significant(p < 0.05),while for the Cleaved caspase-3,the JWLX-H,JWLX-M,and MTX groups could upregulate its protein expression significantly,and the difference was statistically significant(all p < 0.001).Conclusion1.1.Clinical trials showed that the expression of mi R-203a-3p,JAK2,and STAT3 m RNA was upregulated and SOCS3 m RNA expression was decreased by JWLX in skin lesions of patients with blood-heat psoriasis;the relationship between SOCS3 and mi R-203a-3p and STAT3 m RNA were negative.JWLX may enhance the inhibition of JAK2/STAT3 by upregulating the level of SOCS3 m RNA,and improve the PASI score and DLQI score of psoriasis patients with blood heat evidence;and has a high safety profile.2.Animal experiments suggest that JWLX can down-regulate the positive rate of Ki-67,an indicator of epidermal proliferation,and up-regulate AI index,an indicator of apoptosis,in psoriasis-like mice;the PASI score of mice is positively correlated with the positive rate of Ki-67 and negatively correlated with AI index,suggesting that JWLX may reduce the PASI score and Baker score of psoriasis-like mice by regulating the balance of epidermal proliferation/apoptosis.3.JWLX may regulate the mitochondrial apoptosis mediated by this pathway by down-regulating mi R-203a-3p expression and up-regulating SOCS3 expression in skin lesions of psoriasis-like mice,thereby enhancing the inhibition of the JAK2/STAT3 pathway to treat psoriasis.4.JWLX can play a role in the treatment of psoriasis by decreasing the level of anti-apoptotic factor Bcl-2,up-regulating the expression of pro-apoptotic factors Bax,Cyt-c,Caspase-3 and its activated form Cleaved caspase-3,thus inducing mitochondrial apoptosis in mouse epidermal keratinocytes. |
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