失笑散“异病同治”原发性痛经和慢性萎缩性胃炎的作用机制预测及动物试验验证

Prediction and validation of mechanism of action of Shixiao powder in treating primary dysmenorrhea and chronic atrophic gastritis based on“treating different diseases by the same method”

目的了解失笑散治疗原发性痛经(PD)和慢性萎缩性胃炎(CAG)“异病同治”的作用机制。方法采用TCMSP、BATMAN-TCM数据库筛选失笑散有效活性成分及作用靶点,采用Gene Card、Drung Bank、OMIM数据库获取PD和CAG相关蛋白,并取药物、疾病交集靶点制作韦恩图获取失笑散作用靶点与PD、CAG疾病靶点的交集靶点。采用Cytoscape3.9.1软件制作失笑散治疗PD和CAG的成分—靶标网络图,通过String平台构建药物—疾病靶蛋白PPI网络,获取失笑散治疗PD和CAG的核心活性成分、核心靶点。利用DAVID数据库对核心靶点进行GO、KEGG通路富集分析。然后取核心活性成分和核心靶点,通过Autodock Vina软件进行分子对接。大鼠随机分为失笑散低、中、高剂量组(2.5 g/kg,5 g/kg,10 g/kg)、模型组、阳性对照组(布洛芬20 mg/kg)及阴性对照组。除外阴性对照组,其余各组均建立PD动物模型;造模过程中从第4天失笑散低、中、高剂量组灌胃对应治疗剂量的失笑散,阳性对照组灌胃布洛芬20 mg/kg,对照组灌胃等量生理盐水,每天1次。除对照组外,其余各组大鼠腹腔注射缩宫素1.0 m L/kg,对照组皮下注射生理盐水。用药前及末次用药后1 h记录大鼠扭体反应次数,测算子宫收缩抑制率和子宫指数,采用ELISA法检测血清TNF-α、IL-6、IL-1;第12天处死大鼠取子宫组织,采用ELISA法检测VEGFA、PTGS2。结果筛选到失笑散药物有效活性成分15个,失笑散-PD-CAG交集靶点共35个。槲皮素、山柰酚、β-谷甾醇、花生四烯酸、异鼠李素等可能是失笑散治疗PD和CAG的核心有效成分,IL-6、TNF、TP53、VEGFA等可能是失笑散治疗PD和CAG的核心靶点。共获得442条GO生物过程和121条相关信号通路,其中TNF、IL-17等信号通路与失笑散对PD和CAG起治疗作用密切相关。β-谷甾醇、槲皮素等核心有效成分与TP53、TNF、VEGFA等核心靶点对接结果良好。失笑散可降低原发性痛经大鼠子宫指数、扭体次数,升高子宫收缩抑制率(P均<0.05);抑制血清中TNF-α、IL-6、IL-1及子宫组织中PTGS2、VEGFA蛋白的表达,以中高剂量组为著(P均<0.05)。结论失笑散可能通过槲皮素、山柰酚、β-谷甾醇等活性成分与TNF、IL-6、TP53、VEGFA、IL-1β等主要靶点蛋白结合,抑制IL-17、TNF等炎症信号通路治疗PD及CAG,失笑散的用药剂量以5 g/kg、10 g/kg为佳。

Objective To explore the mechanism of action of Shixiao powder in treating primary dysmenorrhea(PD)and chronic atrophic gastritis(CAG)based on“treating different diseases by the same method”.Methods TC-MSP and BATMAN-TCM databases were used to screen the active ingredients and targets of Shixiao powder.PD and CAGrelated proteins were obtained by Gene Card,Drung Bank and OMIM databases.In addition,the intersection targets of drugs and diseases were selected and the Venn diagram was made to obtain the intersection targets of Shixiao powder,PD and CAG.Cytoscape3.9.1 software was used to make the composition-target network diagram of Shixiao powder in the treatment of PD and CAG,and the drug-disease target protein PPI network was constructed by the String platform,so as to obtain the core active ingredients and core targets of Shixiao powder in the treatment of PD and CAG.GO and KEGG path-way enrichment analysis was performed for core targets using DAVID database.Then,the core active ingredients and core targets were selected for molecular docking through Autodock Vina software.The rats were randomly divided into the low-,medium-and high-dose groups(2.5,5,and 10 g/kg),model group,positive control group(ibuprofen 20 mg/kg)and negative control group.Except the negative control group,PD animal model was established in the other groups.In the process of modeling,the rats in the low-,medium-and high-dose groups of Shixiao powder on the fourth day were given intragastric administration of Shixiao powder with corresponding therapeutic dose;the rats in the positive control group were given intragastric administration of 20 mg/kg ibuprofen;the rats in the control group were given intragastric adminis-tration of the same amount of normal saline,once a day.On the 11th day,except the control group,the other groups were intraperitoneally injected with oxytocin 1.0 m L/kg,and the control group was subcutaneously injected with normal saline.The number of twisting response,inhibition rate of uterine contraction and uterine index of rats were measured before med-ication and on the 12th day of medication.Serum tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-1(IL-1)were detected by ELISA.On the 12th day,the rats were sacrificed and uterine tissues were collected.Prosta-glandin-endoperoxide synthase 2(PTGS2)and vascular endothelial growth factor-A(VEGFA)were detected by ELISA.Results A total of 15 active ingredients of Shixiao Powder were screened out,and 35 intersection targets of Shixiao Pow-der-PD-CAG were identified.Quercetin,kaempferol,β-sitosterol,arachidonic acid and isorhamnetin might be the core ef-fective ingredients in the treatment of PD and CAG,while IL-6,TNF,tumor protein P53(TP53)and VEGFA might be the core targets in the treatment of PD and CAG.A total of 442 GO biological processes and 121 related signaling pathways were obtained,among which,TNF,interleukin-17(IL-17)and other signaling pathways were closely related to the thera-peutic effect of Shixiao powder on PD and CAG.The docking results ofβ-sitosterol,quercetin and TP53,TNF,VEGFA and other core active components were good.Shixiao powder could reduce the uterine index and the number of twisting,and increase the inhibition rate of uterine contraction in rats with PD(all P<0.05).The expression levels of TNF-α,IL-6,IL-1 in serum and PTGS2 and VEGFA in uterine tissues were inhibited,especially in the medium-and high-dose Shixiao powder groups(all P<0.05).Conclusions The active ingredients such as quercetin,kaempferol andβ-sitosterol may bind with the main target proteins such as TNF,IL6,TP53,VEGFA and interleukin 1B(IL1B)to inhibit the inflam-matory signaling pathway such as IL-17 and TNF in the treatment of PD and CAG.The optimal dosages of the powder are 5 g/kg and 10 g/kg.