| ObjectiveProfessor Guowei Xuan,the only master of dermatology in China and professor of the Second College of Clinical Medicine of Guangzhou University of Traditional Chinese Medicine,created Yinxie ling tablets by nourishing blood,moistening dryness and removing blood stasis,cooling blood and detoxification and relieving itching.After more than ten years of research,the research team led by Professor Lu Chuanjian streamlined and optimized the prescription to get PSORI-CM02.PSORI-CM02 is composed of traditional Chinese medicine such as Rhizoma Curcumae,Radix Paeoniae Rubra,Herba Sarcandrae,Smilax Glabra and so on.The clinical studies of the research group show that PSORI-CM02 is safe and effective in the treatment of psoriasis vulgaris.Basic studies suggest that PSORI-CM02 can significantly improve the symptoms of psoriasis mice and inhibit the HaCaT prol iferation of human keratinocyte,and has the effects of anti-inflammation and regulating immunity,but the effect of PSORI-CM02 on epidermal autophagy and angiogenesis,which is the key link of psoriasis pathology,needs to be clarified.In this study,the mouse model of psoriasis induced by Imiquimod(IMQ),the human immortalized keratinocyte HaCaT cell model and the IL-17A-induced HUVEC cell model were used to observe the effect of epidermal on epidermal autophagy and angiogenesis,and to explore the regulatory mechanism of PSORI-CM02 on epidermal autophagy and angiogenesis and to further clarify the mechanism and scientific connotation of PSORI-CM02 in the treatment of psoriasis.Methods1.Establish psoriatic mouse model and observe the therapeutic effect of drugs:Balb/c mice were randomly divided into normal control group,IMQ group,Methotrexate(MTX)group,low and high dose groups of PSORI-CM02.The psoriasis model was established by smearing the back skin of mice with IMQ ointment for 7 days.The scores of erythema,scale,psoriasis areas and severity index(PASI)of mice were measured and evaluated every other day.On the seventh day,the samples were photographed and stained with HE.2.The effect of PSORI-CM02 on the expression of autophagy-associated protein and PI3K/Akt/mTOR pathway in psoriatic mouse skin was detected by Western blot,and the expression and distribution of p-mTOR in psoriatic mouse skin were detected by immunohistochemistry.3.Human HaCaT cells were induced by TNF-? to make psoriatic cell model.The cells were divided into four groups:blank control group,TNF-? model group,low and high dose groups of PSORI-CM02,and positive drug group.The proliferation of cells in each group was detected by MTT method,the level of apoptosis was detected by AV-PI flow method,and the activity of Caspase3 in cells was detected by spectrophotometry.4.The expression of autophagy-associated protein mRNA in HaCaT cells was detected by RT-PCR.The expression of autophagy-associated proteins in HaCaT cells was detected by Western blot.The number of autophagy vesicles in HaCaT cells was detected by transmission electron microscope.The expression of Atg16Ll in HaCaT cells was detected by Western blot and cell proliferation was detected by MTT.The effects of autophagy inducer Rapamycin and autophagy inhibitor 3-MA on autophagy and proliferation of HaCaT cells stimulated by TNF-? were analyzed.5.Western blot was used to detect the phosphorylation level of PI3K/Akt/mTOR pathway in HaCaT cells.Western blot was used to detect the expression of Atg16L1 and MTT was used to detect cell proliferation,and the effect of PI3K inhibitor LY294002 on autophagy and proliferation of HaCaT cells stimulated by TNF-? were analyzed.6.Establish psoriatic mouse model:Balb/c mice were randomly divided into normal control group,IMQ group,methotrexate(MTX)group,low and high dose groups of PSORI-CM02.The model of psoriasis was established by smearing the back skin of mice with IMQ ointment for 7 days.On the 7th day of the experiment,the back skin of each group was cut and photographed.The level of oxidative stress molecules in skin tissue was detected by spectrophotometry.The expression of inflammatory cytokines,VEGF and HIF-1? mRNA in skin tissue was detected by fluorescence quantitative PCR,and the expression of VEGFR1,Angiopoietinl and HIF-1? in skin tissue of IMQ mice was detected by immunohistochemistry.Western blot was used to detect the phosphorylation level of MAPK signal pathway in mouse skin.7.The angiogenesis model of human umbilical vein endothelial cell HUVEC induced by IL-17A was established in vitro.The proliferation of HUVEC cells was detected by MTT method,the migration of HUVEC cells was detected by transwell migration assay,the level of oxidative stress molecules in HUVEC cells was detected by spectrophotometry,the expression of inflammatory cytokines mRNA in HUVEC cells was detected by fluorescence quantitative PCR,and the levels of HIF-1? and VEGF in HUVEC cells were detected by ELISA.Western blot was used to detect the expression of angiogenesis molecules and MAPK pathway in HUVEC cells.Results1.The effect of PSORI-CM02 on IMQ-induced psoriasis in mice and its mechanism of regulating autophagyA.The therapeutic effect of PSORI-CM02 on psoriatic miceThe effect on the improvement of animal skin lesions:IMQ could induce psoriasis-like lesions on the back of mice,which was similar to that of human psoriasis,and the PASI score was increased.The low and high doses of MTX and PSORI-CM02 could improve psoriasis-like lesions and reduce PASI scores in mice.The effect on skin histopathology:the results of HE staining showed that the skin of mice in IMQ group showed psoriasis-like histological morphology,and the psoriasis-like histological morphology improved in MTX group and low and high dose groups of PSORI-CM02.B.The effect of PSORI-CM02 on autophagy protein and PI3K/Akt/mTOR pathway in the skin of psoriatic miceThe results of Western blot showed that the expression of autophagy-related protein in IMQ group was significantly lower than that in normal group,the expression of autophagy-related protein in medium and high dose groups was significantly higher than that in normal group.The phosphorylation level of PI3K 85?,Akt and mTOR in IMQ group was significantly increased,and the phosphorylation of PI3K 85?,Akt and mTOR in low and high dose group of PSORI-CM02 could be significantly inhibited.The results of p-mTOR immunohistochemical staining showed that the areas of brown precipitates in the epidermis and dermis of the IMQ model group were significantly increased compared with the normal control group,while the areas of brown precipitates in the epidermis and dermis in the low and high dose groups of PSORI-CM02 and the MTX group were significantly lower than those of the IMQ group.2.Mechanism of PSORI-CM02 on autophagy of HaCaT cells stimulated by TNF-?A.The effect of PSORI-CM02 on proliferation and apoptosis of HaCaT cells stimulated by TNF-?The results of cell proliferation detected by MTT showed that compared with the blank group,the cell proliferation rate of TNT-? group was significantly higher,and compared with TNF-? group,PSORI-CM02 with 0.625mg/ml-10mg/ml significantly inhibited the proliferation of HaCaT cells.According to the results,we chose 2.5mg/ml and 5mg/ml as the concentration of PSORI-CM02 in the follow-up cell experiment.The results of flow cytometry showed that the apoptosis rate of TNF-? group was significantly lower than that of blank group,while that of 2.5mg/ml and 5mg/ml PSORI-CM02 group and MTX group was significantly higher than that of TNF-? group.Compared with the blank control group,the Caspase3 activity of HaCaT cells in TNF-? group had no significant change,but compared with TNF-? group,the Caspase3 activity of HaCaT cells in low and high dose PSORI-CM02 group and MTX group increased significantly.In addition,autophagy inhibitor 3-MA and apoptosis inhibitor Z-VAD can promote cell proliferation in different degrees on the basis of PSORI-CMO2.B.The effect of PSORI-CM02 on autophagy of HaCaT cells stimulated by TNF-?The results of RT-PCR experiment showed that the levels of autophagy-related proteins Beclinl,Atg5,Atg12,Atg7,Atg16L1 and Atg3 mRNA in TNF-? group were significantly lower than those in blank control group,while the levels of Beclinl,Atg5,Atg12,Atg7 and Atg16L1mRNA in HaCaT cells in medium and high dose PSORI-CM02 groups and MTX groups were significantly higher than those in TNF-? group.The results of Western blot assay showed that the expression of autophagy-related proteins Beclinl,Atg5,Atg12,Atg7,Atg16L1 and Atg3 in TNF-? group was significantly lower than that in blank control group,while the protein levels of Beclinl,Atg5,Atg3,Atg12 and Atg16L1 in HaCaT cells in low and high dose PSORI-CM02 groups were significantly higher than those in TNF-? group.The experimental result of transmission electron microscope showed that compared with the blank control group,the number and content of autophagy vesicles in TNF-? group decreased,and compared with TNF-? group,the number of autophagy vesicles in HaCaT cells in low and high dose PSORI-CM02 groups increased.Autophagy inducer Rapamycin was given to HaCaT cells and the effects of PSORI-CM02 combined with autophagy inducer on autophagy and proliferation were observed.The results showed that compared with TNF-? group,PSORI-CM02 combined with autophagy inducer group,autophagy inducer group and PSORI-CM02 group could significantly up-regulate the expression of autophagy protein Atg16L1,and there was no significant difference among the three groups.Compared with TNF-? group,PSORI-CM02 combined with autophagy inducer group,autophagy inducer group and Etretinate optimized prescription group could significantly inhibit the proliferation of HaCaT cells stimulated by TNF-?,and there was no significant difference among the three groups.Autophagy inhibitor 3-MA was given to HaCaT cells and the effects of PSORI-CM02 combined with autophagy inhibitor on autophagy and proliferation were observed.The results showed that compared with TNF-? group,PSORI-CM02 group could significantly up-regulate the expression of autophagy protein Atg16L1.Compared with TNF-?,autophagy inhibitor group,PSORI-CM02 combined with autophagy inhibitor group could significantly inhibit the expression of autophagy protein Atg16L1.And compared with TNF-? group,autophagy inducer group could significantly promote the proliferation of HaCaT cells stimulated by TNF-?.C.The effect of PSORI-CM02 on the regulation of PI3K/Akt/mTOR signal pathway on autophagy of HaCaT cells stimulated by TNF-?The results of Western blot detection showed that the ratio of p-PI3K85a/PI3K85?,p-Akt/Akt and p-mTOR/mTOR in TNF-? group was significantly higher than that in blank control group,while the ratio of p-PI3K85?/PI3K85?,p-Akt/Akt and p-mTOR/mTOR in HaCaT cells in medium and high dose PSORI-CM02 groups was significantly lower than that in TNF-? group.PI3K inhibitor LY294002 was given to HaCaT cells and the effects of PSORI-CMO2 combined with PI3K inhibitor on autophagy and proliferation of cells were observed.The results showed that compared with TNF-? group,PSORI-CMO2 combined with PI3K inhibitor group,PI3K inhibitor group and PSORI-CMO2 group could significantly up-regulate the expression of autophagy protein Atg16L1.ompared with TNF-? group,PSORI-CM02 combined with PI3K inhibitor group,PI3K inhibitor group and PSORI-CM02 group could significantly inhibit the proliferation of HaCaT cells stimulated by TNF-?,and there was no significant difference among the three groups.3.The effect and mechanism of PSORI-CM02 on skin angiogenesisA.The effect and mechanism of PSORI-CM02 on skin angiogenesis in IMQ model miceOn the 7th day of the experiment,the back skin of mice in each group was cut and photographed.The results showed that compared with the normal control group,the number of neovascularization and vascular tortuosity in the IMQ model group increased significantly,while compared with the IMQ model group,the number of neovascularization in the MTX group,the low and high dose of PSORI-CM02 group decreased and the vascular tortuosity improved,The level of oxidative stress molecules in skin tissue was detected by spectrophotometry,and the results showed that compared with the normal control group,the level of SOD in skin tissue of IMQ group was significantly lower,while the levels of LDH,MDA and CAT were significantly higher than those of IMQ group.Compared with the IMQ model group,the level of SOD in skin tissue of high dose PSORI-CM02 group increased significantly,while the levels of LDH,MDA and CAT decreased significantly.The results of RT-PCR test showed that the expression of IL-6,TNF-?,IL-17A and IL-17FmRNA in IMQ group was significantly higher than that in normal control group,while the expression of IL-6,TNF-?,IL-17A and IL-17FmRNA in high dose PSORI-CM02 group was significantly lower than that in IMQ model group.The results of RT-PCR detection showed that the expression of VEGF and HIF-1? mRNA in the skin tissue in IMQ group was significantly higher than that in normal control group,while the expression of VEGF and HIF-1? mRNA in low dose PSORI-CM02 group was significantly lower than that in IMQ model group.The results of immunohistochemical staining of VEGFR1,Angiopoietinl and HIF-1? showed that the color and area of brown precipitates in epidermis and dermis in IMQ model group were significantly higher than those in normal control group,while the color and area of brown precipitates in epidermis in low and high dose PSORI-CM02 group and MTX group were significantly lower than those in IMQ group.The results of Western blot detection showed that compared with the normal control group,the expression of VEGFR1,Angiopoietinl and HIF-1? protein in the skin tissue in IMQ group increased significantly,compared with IMQ model group,the expression levels of VEGFR1,Angiopoietinl and HIF-1? protein in the skin tissue in high dose PSORI-CM02 group and MTX group decreased significantly,and the ratio of p-ERK/ERK,p-P38/P38 and p-JNK/JNK in the skin tissue in IMQ group increased sign if icantly compared with the normal control group.Compared with IMQ model group,the ratio of p-ERK/ERK,p-P38/P38 and p-JNK/JNK in skin tissue in low dose group of PSORI-CM02 and MTX group decreased significantly.B.The effect of PSORI-CM02 on proliferation and migration of HUVEC cells stimulated by IL-17AThe results of cell proliferation detected by MTT method showed that the cell proliferation rate of IL-17A group was significantly higher than that of blank group,and the proliferation of HUVEC cells was significantly inhibited by PSORI-CM02 of 1.25mg/ml-15mg/ml concentration compared with IL-17A group.According to the results,we chose 1.25mg/ml and 2.5mg/ml as the concentration of PSORI-CM02 in the follow-up cell experiment.The results of cell migration assay showed that compared with the blank group,the number of cell migration in the IL-17A group was significantly increased,and compared with the IL-17A group,the number of HUVEC cells in the PSORI-CM02 group at the concentration of 1.25mg/ml and 2.5mg/ml was significantly lower than that in the HUVEC group.The level of oxidative stress molecules in HUVEC cells was detected by spectrophotometry.The results showed that compared with the blank group,the level of SOD in HUVEC cells in IL-17A group was significantly lower,while the levels of LDH,ROS,MDA,GSH and CAT were significantly higher than those in IL-17A group.Compared with the IL-17A group,the level of SOD in the high dose of PSORI-CM02 group increased significantly,while the levels of ROS,GSH,MDA and CAT decreased significantly.The results of RT-PCR detection showed that the levels of IL-1 ?,TNF-? and IL-6 in HUVEC cells in IL-17A group were significantly higher than those in blank group,while the levels of IL-6 in middle dose of PSORI-CM02 group and the levels of TNF-? in high dose of PSORI-CM02 group were significantly lower than those in IL-17A group.The results of ELISA detection showed that the levels of HIF-la and VEGF in HUVEC cells in IL-17A group were significantly higher than those in blank group,while the levels of HIF-1? in high dose of PSORI-CM02 group and the levels of VEGF in medium dose of PSORI-CM02 group were significantly lower than those in IL-17A group.The results of Western blot detection showed that compared with the blank group,the expression levels of VEGFR1,VEGFR2,Angiopoietinl and HIF-1? protein in HUVEC cells in IL-17A group were significantly increased,while the expression levels of VEGFR1,VEGFR2,Angiopoietinl and HIF-1? protein in medium dose group of PSORI-CM02 were significantly lower than those in IL-17A group.Compared with the blank group,the proportion of p-ERK/ERK,p-P38/P38 and p-JNK/JNK in HUVEC cells in IL-17A group increased significantly,while compared with IL-17A group,the proportion of p-ERK/ERK,p-P38/P38 and p-JNK/JNK in low dose group of PSORI-CM02 and MTX group decreased significantly.Conclusion1.In the mouse model of psoriasis induced by IMQ,PSORI-CM02 can up-regulate the expression of autophagy-related proteins in the skin of psoriatic mice by inhibiting the activation of PI3K/Akt/mTOR pathway,so as to improve the psoriatic lesions induced by IMQ.2.In the in vitro model of HaCaT cells stimulated by TNF-?,PSORI-CM02 can up-regulate the autophagy level of HaCaT cells by inhibiting the phosphorylation of PI3K/Akt/mTOR pathway,thereby inhibiting cell proliferation and inducing apoptosis.3.In the IL-17A—induced HUVEC cells model,by inhibiting the phosphorylation of MAPK signal pathway,PSORI—CMO2 can inhibit the synthesis of angiogenesis-related molecules such as VEGFR1,VEGFR2,Angiopoietinl and HIF-la stimulated by IL-17A,inhibit the expression of oxidative stress molecules and inflammatory cytokines,and then inhibit cell migration and proliferation.4.In the mouse model of psoriasis induced by IMQ,PSORI-CM02 can inhibit the activation of MAPK signal pathway,inhibit the expression of oxidative stress molecules and inflammatory cytokines,inhibit the synthesis of angiogenesis-related molecules in mouse skin,and then inhibit skin neovascularization. |
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