| Objective:This study observed the clinical efficacy of Wen’s Machixian Decoction in treating acute eczema patients with the syndrome of damp heat accumulating skin through a randomized controlled trial,and explored the possible mechanism of Wen’s Machixian Decoction in treating acute eczema through animal experiments,and whether it could alleviate the inflammatory reaction in vivo through the“microorganism-intestine-skin” axis,thereby providing scientific basis for the clinical application of Wen’s Machixian Decoction in treating acute eczema.Methods:Clinical research: In this study,we randomly recruited 90 acute eczema patients with the syndrome of damp heat accumulating skin and randomly divided them into the treatment group(received oral Wen’s Machixian Decoction combined with topical 3% boric acid solution)and control group(received oral Longdan Xiegan Pill combined with topical 3% boric acid solution),with 45 patients in each group.The course of treatment of both groups was 2 weeks.Eczema Area and Severity Index(EASI),Pruritus Degree and TCM Syndrome were scored at the 0th week and the 2th week after enrollment.The safety of Wen’s Machixian Decoction was evaluated by adverse events during treatment,and the recurrence was observed after 1 month of follow-up after the treatment to evaluate the long-term efficacy or adverse reactions of the drug.Peripheral venous blood of all patients was drawn before and after the treatment,and the content of IFN-γ,TNF-α,IL-4 were detected by ELISA.Animal experiment: 36 BALB/c male mice were randomly divided into control group(no modeling and no administration),model group,Longdan Xiegan Pill group,Wen’s Machixian Decoction high dose group,Wen’s Machixian Decoction medium dose group,and Wen’s Machixian Decoction low dose group,with 6 mice in each group.Except for the control group,the rest groups used acetone-olive oil of 2,4-dinitrochlorophenylacetone(DNFB)to make the model on the back and right ear of mice.After successful modeling,the drug groups were administered continuously for 7 days,and the mice in the control group and model group were given the same amount of normal saline.At the end of the experiment,the erythema area,edema degree,epidermis peeling and scabbing of the right auricle skin lesions of mice were scored.After the mice were killed,the pathological changes of the right auricle lesions were observed by Hematoxylin-eosin(HE)staining,detection of IFN-γ,TNF-α,IL-4 content in serum of mice in each group by ELISA,and detection of changes in skin microbiota and gut microbiota by 16 S rRNA gene high-throughput sequencing technology.HE staining was used to observe the pathological histology of ileum of mice in each group.RT-PCR was used to detect the expression of tight junction protein ZO-1 and Occludin genes in ileum tissues of mice in each group.The content of sIgA in feces of mice in each group was detected by ELISA.Results:Clinical research:(1)Comparison of study completion and baseline data: A total of 90 acute eczema subjects with the syndrome of damp heat accumulating skin were recruited,of which 5 cases were exfoliated(2 cases in the treatment group and 3 cases in the control group),and 85 cases were actually completed.There was no statistical difference between the two groups in the completion and abscission of cases(P>0.05),which was comparable.There was no statistical difference between the two groups in demographic data,disease characteristics and other general data(P>0.05),which was comparable.(2)EASI score: The intra group comparison showed that the EASI score of subjects in both groups decreased significantly after treatment compared with those before treatment(P<0.01).The comparison between groups indicated that the treatment group was better than the control group in reducing the EASI score of acute eczema(P<0.05).(3)Pruritus score: The intra group comparison showed that the score of pruritus degree in the treatment group and the control group decreased significantly after treatment compared with those before treatment(P<0.01).The comparison between the groups showed that the effect of the treatment group and the control group in improving the itching degree of patients with acute eczema was equivalent(P>0.05).(4)TCM Syndrome score: The intra group comparison indicated that the score of TCM syndromes in both groups decreased significantly after the treatment(P<0.01).The comparison between groups indicated that the improvement effect of TCM syndrome score in the treatment group was similar to that in the control group(P>0.05).(5)Total score: The intra group comparison showed that the total score of subjects in both groups decreased significantly after the treatment compared with those before the treatment(P<0.01).The comparison between groups showed that the improvement of the total score of the treatment group was better than that of the control group(P<0.05).(6)Clinical efficacy: The clinical efficacy was evaluated according to the difference between the total score of the two groups before and after treatment.The results showed that the total clinical effective rate of the treatment group was significantly better than that of the control group(P<0.05).(7)Safety evaluation: There was no significant difference in the incidence of adverse events between the treatment group and the control group(P>0.05).(8)Recurrence evaluation: There was statistical difference in the relapse rate between the two groups(P<0.05),indicating that the treatment group could better prolong the curative effect and reduce the recurrence rate than the control group.(9)Content of IFN-γ,TNF-α,IL-4 in peripheral blood: The intra group comparison showed that both groups could increase IFN-γ level after treatment compared with that before treatment(P<0.05),and reduce TNF-α and IL-4 level(P<0.05),indicating the serum inflammatory factor IFN-γ,TNF-α,IL-4 could be effectively adjusted.The comparison between groups reflected that the treatment group and the control group had no statistical difference in improvement of IFN-γ,TNF-α,IL-4(P>0.05).Animal experiment:(1)Mice auricle lesion score in each group: Compared with the control group,the mice in rest groups after the successful modeling showed obvious redness,swelling,papule and scabbing in the right ear compared with the left ear,similar to human eczema like skin lesions.On the 7th day of the experiment,the EASI score of the right ear skin lesions was significantly increased(P<0.01).Compared with the model group,the EASI scores of the right ear lesions in each treatment group were significantly lower(P<0.01).Compared with the Longdan Xiegan Pill group,the EASI score of the right ear skin lesions in the high dose group and the middle dose group of Wen’s Machixian Decoction decreased significantly(P<0.01),and the middle dose group of Wen’s Machixian Decoction decreased most significantly(P<0.01).(2)Histopathological changes in the right ear of mice in each group: The epidermal structure of mice in the control group was intact without obvious pathological changes.The skin of the right ear of mice in the model group showed various pathological changes such as thickening of the epidermis,increasing of the number of acanthocytes,increasing of the volume of acanthocytes,varying degrees of necrosis of collagen fibers in the dermis,disappearance of collagen fiber structure,infiltration of inflammatory cells,and proliferation of fibrous tissue.Interestingly,the above pathological changes were significantly alleviated after taking high and medium doses of Wen’s Machixian Decoction.(3)Content changes of IFN-γ,TNF-α and IL-4 in peripheral blood of mice in each group: Compared with the control group,the content of TNF-α and IL-4 in serum of mice in other groups increased(P<0.05),while the content of IFN-γ decreased(except for the medium dose group of Wen’s Machixian Decoction,P<0.05).Compared with the model group,the content of TNF-α and IL-4 in serum of mice in all medication administration groups decreased to some extent,while the content of IFN-γ increased in varying degrees.The content of TNF-α in high and medium dose groups of Wen’s Machixian Decoction decreased significantly(P<0.01).The content of IFN-γ in the medium dose group of Wen’s Machixian Decoction increased significantly(P<0.05)and IL-4 decreased significantly(P<0.01).(4)Changes of skin microbiota of mice in each group: Compared with the control group,Observed＿species index and Chao1 index in the model group showed a decreasing trend,while Simpson index and Shannon index showed a decreasing trend.Taking high dose of Wen’s Machixian Decoction could reduce Observed＿species index,Chao1 index,and increase Simpson index and Shannon index,but there was no statistical difference(P>0.05).Compared with the model group,after taking the medium dose of Wen’s Machixian Decoction,the skin microbiota was closest to that of the healthy mice,and the proportion of Firmicutes,Bacilli,Bacillales,Staphylococcaceae decreased,while the proportion of Proteobateria,Bacteroidetes,Gammaproteobartia,Bacteroidia,Clostridiales,Burkholderiaceae increased.At the generic level,each dose of Wen’s Machixian Decoction could reduce the relative abundance of Staphylococcus,Acinetobacter,Lactobacillus,Ralstonia and Pseudomonas.(5)The correlation of skin differential genus and serum inflammatory factor(IFN-γ,TNF-α,IL-4): In the Longdan Xiegan Pill group,the relative abundance of Streptococcus,Uclassified＿Lachnospiraceae,Lachnospiraceae NK4A136 group,Pantoea was correlated with the levels of IL-4.In the Wen’ s Machixian Decoction high dose group,the relative abundance of Helicobacter,Cetobacterium,Janthinobacterium,Stenotrophomonas was correlated with the levels of IFN-γ.In Wen’s Machixian Decoction medium dose group,the relative abundance of Bacteroides,Janthinobacterium was correlated with the levels of IFN-γ,and the relative abundance of Blautia was correlated with the levels of IL-4.P<0.05.(6)Histopathological changes of ileum in mice of each group: No pathological changes such as mucosal degeneration,necrosis and inflammatory cell infiltration were found in the control group.Pathological changes were observed in the model group,but the overall pathological changes were mild.In the model group,the mucosal layer and submucosa structure of ileum tissue were slightly damaged,and local mucosal layer was degenerated and necrotic,with pyknosis or even disintegration of cell nucleus;cytoplasm dissolution was empty and reticular;part of intestinal villi fell off;intestinal gland lost its normal structure,with a small amount of inflammatory cell infiltration,and part of it involved the submucosa.In the high and medium dose groups of Wen’s Machixian Decoction,the above pathological changes were alleviated to varying degrees.(7)Expression of tight junction protein gene in ileum of mice in each group:Although there was no statistical difference in the expression of ZO-1 m RNA between groups(P>0.05),the expression of ZO-1 m RNA in each group showed a downward trend compared with the control group.Compared with the model group,the expression of ZO-1 m RNA in Longdan Xiegan Pill group and Wen’s Machixian Decoction medium dose group increased,and Wen’s Machixian Decoction medium dose group increased the most.Compared with the control group,the expression of Occludin m RNA in each group decreased significantly(P<0.01).Compared with the model group,the expression of Occludin m RNA in the high and middle dose groups of Wen’s Machixian Decoction increased,but there was no statistical difference(P>0.05).(8)Fecal s Ig A content of mice in each group: Compared with the control group,the content of s Ig A in the model group decreased significantly(P<0.01).Compared with the model group,the content of s Ig A in all medication administration groups was significantly increased(P<0.01),among which the high dose group of Wen’s Machixian Decoction was the most significant.(9)Changes of gut microbiota of mice in each group: Observed＿species index,Chao1 index,Simpson index and Shannon index in the model group showed a decreasing trend compared with the control group.The above indexes could be increased after taking Wen’s Machixian Decoction,especially in the middle dose.Wen’s Machixian Decoction could inhibit the production of Deferribacteres,Proteobacteria,Deferribacterales,Campylobacterales,Alistipes,Odoribacter,Mucispirillum,Helicobacter,Rikenellaceae RC9 gut group,Millionella,Ruminiclostridium 5,Ruminiclostridium 6.(10)The correlation of gut differential genus and serum inflammatory factor(IFN-γ,TNF-α,IL-4): In the Longdan Xiegan Pill group,the relative abundance of Enterorhabdus,Millionella,Mucispirillum,Helicobacter,Lactobacillus,Uclassified＿Lachnospiraceae was correlated with the levels of TNF-α;the relative abundance of Lachnospiraceae UCG-006 was correlated with the levels of IL-4.In the Wen’s Machixian Decoction high dose group,the relative abundance of Blautia,Ruminiclostridium 9,Ruminococcaceae UCG-014,Uclassified＿Desulfovibrionaceae was correlated with the levels of IFN-γ;the relative abundance of Uclassified＿Peptococcaceae was correlated with the levels of TNF-α.In the Wen’s Machixian Decoction medium dose group,the relative abundance of Rikenellaceae RC9 gut group,Lactobacillus,Lachnoclostridium,Lachnospiraceae FCS020 group,Lachnospiraceae UCG-006,Ruminiclostridium was correlated with the levels of IFN-γ;the relative abundance of Blautia,Desulfovibrio was correlated with the levels of TNF-α;the relative abundance of Ruminiclostridium 5 was correlated with the levels of IL-4.In the Wen’s Machixian Decoction low dose group,the relative abundance of Bacteroides was correlated with the levels of IFN-γ.P<0.05.Conclusion:(1)Wen’s Machixian Decoction could effectively reduce the EASI score,Pruritus Degree,TCM Syndrome score and total score of acute eczema patients with the syndrome of damp heat accumulating skin,thereby improving the overall clinical effectiveness and safety.(2)The level of IFN-γ in patients and mice with acute eczema after treatment with Wen’s Machixian Decoction increased,and the level of TNF-α,IL-4 decreased,thereby reducing inflammatory reaction and achieving therapeutic goal.(3)Wen’s Machixian Decoction could regulate the imbalance of skin microbiota in mice with acute eczema,and the abundance of skin differential genus had a stronger correlation with inflammatory factors(IFN-γ,TNF-α,IL-4)in the high-dose group of Wen’s Machixian Decoction,indicating that differential bacteria could better indicate changes in inflammatory factors(IFN-γ,TNF-α,IL-4)at high doses.(4)Wen’s Machixian Decoction could regulate gut microbiota,repair the intestinal barrier damage,and the abundance of intestinal differential genus had a stronger correlation with inflammatory factors(IFN-γ,TNF-α,IL-4)in the group of Wen’s Machixian Decoction.Thus,Wen’s Machixian Decoction might mediate the “intestine-skin” axis by regulating the “intestine-skin microbiota”,and regulate the the level of IFN-γ,TNF-α,IL-4 to affect the overall homeostasis of the “intestine-skin” axis,thereby playing a role in the treatment of acute eczema. |
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