| BackgroundPsoriasis is a common chronic inflammatory recurrent skin disease,mainly characterized as erythema and scales.Psoriasis is also called as "Bai Bi" in traditional Chinese medicine,which was mostly caused by the loss of blood in the camp,blood heat content,wind and dryness,and skin deprivation.At present,the differentiation of syndromes of psoriasis is mostly based on "blood separation",and the blood-heat syndrome is the most common syndrome type in psoriasis.Qingre Liangxue Decoction is derived from the classic famous prescription "Xijiao Dihuang Decoction" and the instructor's 30 years' clinical experience.Preliminary clinical research and basic experiments have confirmed the safety and efficacy of Qingre Liangxue Decoction in treating psoriasis through various biological mechanisms.The treatment of diseases with traditional Chinese medicine compound has the characteristics of multiple components,multiple targets,and multiple pathways.The effective components and mechanisms of Qingre Liangxue Decoction for psoriasis have not been elucidated.Network pharmacology is a discipline which uses modern bioinformatics technology to reveal the network regulation of traditional Chinese medicine on the body based on multiple public databases such as drug molecules,genes,and proteins.Therefore,network pharmacology could be applied to explain the "component-target" network of Chinese herbal medicines for treating diseases.Regulating metabolism may be a new idea for the treatment of psoriasis.Glucose Transporter 1(GLUT1)is a widely expressed glucose transporter,which may participate in the pathogenesis of psoriasis by promoting epidermal proliferation,activating inflammation and angiogenesis and other mechanisms.GLUT1 is regulated by the PI3K/AKT signaling pathway.At present,there is no research to investigate whether Qingre Liangxue Decoction and its active ingredients may treat psoriasis through the PI3K/AKT/GLUT1 signaling pathway.Objectives1.To explore the possible bioactive components and targets of Qingre Liangxue Decoction in the treatment of psoriasis by using network pharmacology.2.To compare the expression of GLUT1 protein in the skin lesions of blood-heat psoriasis patients and the skin of healthy controls.3.To observe whether quercetin,the main active ingredient of Qingre Liangxue Decoction,plays a therapeutic effect on psoriasis through regulating PI3K/AKT/GLUT1 signaling pathway by in vivo and in vitro experiments.MethodsStudy One:Through the database mining and literature search,the molecular component database of Qingre Liangxue Decoction was constructed.And the computer-based method was used to obtain the effective chemical composition of Qingre Liangxue Decoction by evaluating the ADME properties of the drug molecules,and the component-target network was further constructed.And protein-protein interaction,GOBP analysis and Reactome pathway analysis were used to reveal the possible mechanism of Qingre Liangxue Decoction in the treatment of psoriasis.Study Two:20 patients with blood-heat psoriasis vulgaris and 15 healthy controls were enrolled.Immunohistochemical technique was used to detect the expression of GLUT1 protein in skin lesions of blood-heat psoriasis patients and to compare the differences between the two groups.Study Three:47 BALB/c male mice aged 6 to 8 weeks were selected and randomly divided into 6 groups,7 in the blank control group,and 8 in each of the remaining groups.Except for the blank group,the remaining groups were applied to the hair removal back with 5%imiquimod cream,once a day,62.5 mg each time for 7 days.Tripterygium wilfordii group were administered tripterygium wilfordii polyglycosidic solution with concentration of 7.56 mg/kg by gavage.Low-dose,middle-dose and high-dose quercetin group were administered quercetin solution with concentration of 30 mg/kg,60 mg/kg and 120 mg/kg by gavage.Blank control group and model group were administered isosmotic normal saline by gavage.After 7 days of intervention,the skin in back lesions and serum of mice were collected on the 8th day.The changes of skin lesions through the naked eye were observed and PASI scores were performed.HE staining was conducted to compare the pathological changes of skin tissues and the changes in epidermal thickness were measured.Immunohistochemical technique was used to detect the expression of GLUT1 protein in mouse lesions.Quantitative Real-time PCR was used to detect the relative expression levels of AKT mRNA and GLUT1 mRNA in skin lesions of mice.Western blot was used to detect the expression levels of AKT,pAKT and GLUT1 protein in skin lesions of mice and flow cytometry was used to detect the content of serum inflammatory factors in mice.Study Four:HaCaT cell was divided into 9 groups,including blank control group(Control),simulated psoriasis disease group(Model A),disease+PI3K/AKT pathway inhibition group(Model B),disease+high-dose Quercetin group(Model A+QCH),disease+middle-dose quercetin group(Model A+QCM),disease+low-dose quercetin group(Model A+QCL),and disease+inhibition+high-dose quercetin Group(Model B+QCH),disease+ inhibition+medium-dose quercetin group(Model B+QCM)and disease+inhibition+low-dose quercetin group(Model B+QCL).CCK-8 method was used to detect the effect of quercetin on the proliferation of HaCaT cells.Flow cytometry was used to detect the effect of quercetin on apoptosis of HaCaT cells.Western blot was used to detect the expression of AKT,pAKT and GLUT 1 proteins.ResultsStudy One:Based on database construction and computer analysis,we obtained 131 active molecules and 483 targets of Qingre Liangxue Decoction.According to Degree ranking,quercetin may be the main active ingredient in Qingre Liangxue Decoction,and its main targets involved PI3K/AKT signaling pathway.Qingre Liangxue Decoction treated psoriasis through many biological processes,including regulating metabolism and immunity.Study Two:GLUT1 was strongly expressed in the cell membrane of some spine cells and basal cells,and weakly expressed in the nucleus of some spine cells and basal cells.The expression levels of GLUT1 in the spine cells and basal cell membranes of blood-heat psoriasis patients were significantly higher than those in healthy controls(P<0.001).Study Three1.In terms of skin lesions,the mice in the model group showed typical "psoriasis-like"changes such as erythema,hypertrophy,and scale.The erythema,hypertrophy,and scale in the quercetin group were less severe than those in the model group at the same time point.In terms of PASI score,the total score,erythema,hypertrophy,and scale scores on the 8th day of the quercetin group were significantly lower than those of the model group(all P values<0.05).2.In terms of pathological changes,the mice in the model group showed typical "psoriatic-like" changes.The hyperkeratosis of the epidermis and the hypertrophy of the spinous layer in each dose group of quercetin were less than those in the model group.In terms of epidermal thickness,the model group was significantly higher than the blank control group(130.93 ±69.66 ?m vs 21.81 ± 2.84 ?m,P<0.001).Moreover,the epidermal thickness of tripterygium wilfordii group,low-dose quercetin,medium-dose quercetin,and high-dose quercetin groups were 77.93 ± 12.54 ?m,71.87 ± 12.99?m,77.73 ±10.08 ?m,62.33 ± 11.41?m in order,and were significantly lower than the model group(all P values<0.05).3.Immunohistochemical results showed that the GLUT1 protein expression level of the model group was significantly higher than that of the blank control group(P<0.001),and the GLUT1 protein expression level of the tripterygium wilfordii group,low-dose quercetin,medium-dose quercetin,and high-dose quercetin groups were significantly lower than that of the model group(all P values<0.001).4.The results of quantitative Real-time PCR showed that the relative expression levels of AKT mRNA and GLUT1 mRNA in the skin lesions of the model group were significantly higher than that of the blank control group(all P values<0.001),and the relative expression levels of AKT mRNA and GLUT1 mRNA in the remaining groups were was significantly lower than the model group(all P values<0.001).The relative expression levels of AKT mRNA and GLUT1 mRNA in low-dose,medium-dose,and high-dose quercetin groups were in descending order.5.Western blot results showed that the ratio(pAKT/AKT)of relative expression level of pAKT to AKT protein in the skin lesions of the model group was significantly higher than that in the control group(P<0.05).The pAKT/AKT in the tripterygium wilfordii and high-dose quercetin group were significantly lower than that of the model group(P<0.05),and the pAKT/AKT of the low-dose and middle-dose quercetin groups were lower than that in the model group,but the difference were not statistically significant(all P values>0.05).The relative expression of GLUT1 protein in the model group was significantly higher than that in the control group(P<0.001).The relative expression of GLUT1 protein in the tripterygium wilfordii,middle-dose and high-dose quercetin groups were significantly lower than that in the model group(all P values<0.05).The relative expression of GLUT1 protein in the low-dose quercetin group was lower than that in the model group but there was no significant statistical difference(P>0.05).At the same time,the relative expression level of GLUT1 in the low-dose,middle-dose and high-dose quercetin groups were decreased sequentially.6.In terms of serum inflammatory factors,the levels of TNF-?,IL-17A,IL-6,and IL-10 in the model group were significantly higher than that in the blank control group(all P values<0.05).Although the IFN-y,IL-21,IL-22 levels in the model group were also higher than that in the blank control group,the differences were not statistically significant(all P values>0.05).At the same time,the levels of TNF-?,IL-6 and IL-17A in the high-dose quercetin group were significantly lower than those in the model group(all P values<0.05).Study Four1.The results of CCK-8 method showed that quercetin could inhibit the proliferation of HaCaT cells.The IC50 of quercetin for inhibiting HaCaT cells at different administration times were:24h,IC50=160.3 mM,48h,IC50=73.28 mM,72h IC50=42.20 mM.2.The results of apoptosis showed that the apoptosis rate in Model A group was significantly lower than that in blank control group(P<0.05),and the apoptosis rate in Model B group was significantly higher than that in blank control group(P<0.001).The apoptosis rate in Model A+QCH,Model A+QCM and Model A+QCL groups were gradually decreased and were significantly higher than that in Model A group(all P values<0.001).The apoptosis rate of Model B+QCH and Model B+QCM were gradually decreased and were significantly higher than that of Model B group(all P values<0.001).The apoptosis rate of Model B+QCL group was also higher than that of Model B group,but the difference was statistical significance(P>0.05).3.Western blot results showed that the pAKT/AKT in Model A group was significantly higher than that in Control group(P<0.001).The relative expression of GLUT1 protein in Model A group was significantly higher than that in Control group(P<0.001).The pAKT/AKT in the Model A+QCH group,Model A+QCM group and Model A+QCL group were increased and were significantly lower than those in Model A group(all P values<0.001).The relative expression of GLUT1 protein in Model A+QCH group was significantly lower than that in Model A group(P<0.05).The relative expression of GLUT1 protein in Model A+QCM group and Model A+QCL group was also lower than that in Model A group,but the difference was not statistically significant(P>0.05).The pAKT/AKT in the Model B+QCH group and Model B+QCM group were increased and were significantly lower than that in Model B group(all P values<0.01).The relative expression levels of GLUT1 protein in Model B+QCH group and Model B+QCM group were increased and were significantly lower than those in Model B group(P<0.01,P<0.05).The relative expression of GLUT1 protein in Model B+QCL group was also lower than that in Model B group,but the difference was not statistically significant(P>0.05).Conclusions1.Qingre Liangxue Decoction had a multi-component and multi-target network effect in the treatment of psoriasis.Quercetin may be the main active ingredient of Qingre Liangxue Decoction,and its mechanism may be related to PI3K/AKT signaling pathway.2.Quercetin may play a therapeutic role in psoriasis by regulating the PI3K/AKT/GLUT1 signaling pathway. |
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