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ObjectiveTo preliminarily predict the targets and signaling pathways of indole-3-methanol in the treatment of obesity based on molecular docking technology and network pharmacology， and then verify the prediction results by the experiment in vitro. MethodThe pharmacological targets of indole-3-methanol were obtained from SwissTargetPrediction and literature review. Obesity-related targets were obtained from Online Mendelian Inheritance in Man （OMIM）， GeneCards， and Comparative Toxicogenomics Database （CTD）. The protein-protein interaction network of the targets of indole-3-methanol and obesity was built by STRING. Cytoscape 3.8.2 was used for target screening. Gene ontology （GO） functional annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment were performed for the common targets shared by obesity and indole-3-methanol in DAVID 6.8. AutoDock Vina 1.1.2 was employed to perform the molecular docking between indole-3-methanol and disease targets. Finally， the in vitro experiment was carried out to verify the anti-obesity effect of indole-3-methanol. ResultIndole-3-methanol and obesity shared 80 common targets， which included matrix metalloproteinase （MMP）-9， Janus kinase （JAK） 2， etc. KEGG enrichment predicted that indole-3-methanol mainly acted on tumor necrosis factor （TNF）， vascular endothelial growth factor （VEGF）， tyrosine kinase receptor 2 （ErbB2）， and epidermal growth factor receptor （EGFR） signaling pathways in the treatment of obesity. Molecular docking showed that indole-3-methanol had good binding activity with fat mass and obesity-associated protein （FTO）. The results of Western blot， MTT assay， and oil-red O staining showed that indole-3-methanol down-regulated the expression of FTO in 3T3-L1 cells （P<0.05）. ConclusionIndole-3-methanol may treat obesity by down-regulating the expression of FTO protein and further inhibiting adipocyte proliferation. This study provides an experimental basis for deciphering the anti-obesity mechanism of indole-3-methanol.
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Objective To predict the potential mechanism of Punicalagin in the treatment of Inflammatory bowel disease by network pharmacology.Methods The intersection genes of Punicalagin and IBD were obtained from the database,and PPI,GO and KEGG pathways were enriched and analyzed.Punicalagin and the target were verified by molecular docking.C57BL/6J mice were drunk dextran sulfate sodium to establish inflammatory enteritis model,and were given Punicalagin for 7 d of intervention.During the administration,signs of mice in each group were observed,daily disease activity index was calculated;Intestinal permeability test after administration;The colon tissue was stained with hematoxylin eosin to observe the pathological changes and calculate the histological damage score;Detection of tumor necrosis factor(TNF-α),Interleukin-10(IL-10),myeloperoxidase(MPO),chemokine 1(CXCL1)and other cytokines in colon tissue of mice by ELISA.Detection of TNF-α,IL-6,MPO and CXCL1 level in mouse serum by ELISA.CCK8 method was used to determine the effect of Punicalagin on the proliferation activity of caco-2 cells.The levels of cytokines released by caco-2 cells induced by lipopolysaccharide(LPS)were detected by ELISA.Results 14 common targets of Punicalagin and IBD were obtained,including tumor necrosis factor(TNF),arachidonic acid-5-lipoxygenase(ALOX5)and vascular endothelial growth factor A(VEGFA).KEGG enrichment analysis predicted that the treatment of IBD by Punicalagin mainly acted on arachidonic acid signaling pathway,age-rage signaling pathway,VEGR signaling pathway and Ras signaling pathway.Molecular docking showed that Punicalagin had good docking activity with TNF receptor.Compared with the model group,the decreasing range of body mass in Punicalagin group abated(P<0.01);the disease activity index of Punicalagin group decreased significantly(P<0.01);The congestion and edema of colonic mucosa were significantly reduced,and the histological injury score was significantly reduced(P<0.01);The level of TNF-α,IL-1β,MPO,CXCL1,IL-6,IL-18,IFN-γ in colon tissue was significantly decreased(P<0.01);20-300 μmol·L-1 Punicalagin promoted caco-2 cell proliferation and inhibited TNF-α secretion induced by LPS,up-regulation of IL-10 levels.Conclusion Punicalagin inhibits the secretion of TNF-α and other proinflammatory factors,up-regulation of the level of anti-inflammatory factor IL-10,and improvement of colonic inflammatory response in IBD mice.
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OBJECTIVE：To st udy the inhibitory mechanism of pomegranate peel polyphenols （PPP）on the proliferation of human prostate cancer PC 3 cells based on autophagy and apoptosis pathway. METHODS ：CCK-8 assay was used to investigate the effects of PPP with different concentrations （25-300 μg/mL）on PC 3 cell activity after culturing for 24，48，72 h，so as to screen the drug concentration and treatment time. PC 3 cells were divided into control group （complete culture medium ），PPP low- ，medium- and high-concentration groups. After treated for 48 h，flow cytometry and Annexin V-FITC/PI staining were used to detect cell cycle distribution and apoptosis of PC 3 cells. Western blotting assay was used to detect the expression of apoptosis-related protein as Bax ，Bcl-2，as well as the expression of autophagy-related proteins as LC 3，Beclin-1，p62，Atg12 and Atg 16. RESULTS ：The culturing time was chosen as 48 h. IC 50 of PPP was 110 μg/mL，and 50，100，200 μg/mL were chosen as low，medium，high concentrations of PPP. Compared with control group ，the percentage of PC 3 cells at phase G 0/G1 decreased significantly in PPP low- and medium-concentration groups while increased significantly at phase S ；that of PC 3 cells at phase G 0/G1 increased significantly in PPP high-concentration group ； while that of PC 3 cells at phase G 2/M decreased significantly in PPP medium- and high-concentration groups （P＜0.05 or P＜0.01）. The apoptosis rate of PC 3 cells was increased significantly in PPP groups （P＜ 0.05 or P＜0.01）. Compared with control group ，protein expression of anti-apoptosis protein Bcl- 2 and autophagy-related promoting protein p 62 were decreased in PPP groups to different extents ，while protein expression of promoting-apoptosis protein Bax as wells as autophagy-related protein LC 3-Ⅱ/LC3-Ⅰ ration and protein expression of Beclin- 1，Atg5，Atg12 and Atg 16 were increased to different extents ；there was statistical significance in above indexes in PPP high-concentration group and some of above indexes in PPP low- and medium-concentration groups （P＜0.05 or P＜0.01）. CONCLUSIONS ：PPP can inhibit the proliferation of human prostate cancer PC 3 cells，mechanism of which may be related to inducing autophagy and promoting apoptosis.
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There are many disadvantages in the traditional teaching of pharmacology experiment, so it is difficult to assess students' experimental skills and comprehensive levels. Based on the comprehen-siveness, fairness, objectivity and reciprocity, we put forward a test-system which contains experimental preparation oral, experimental skills, experimental records, experimental reports, design experiments, ordi-nary performance and final experiment theory exam, hoping to provide a reference for the establishment of a new pharmacology experiment course assessment system.
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AIM To research the therapeutic effects of Hanbi Formula (Astragali Radix,Aconiti Radix cocta,Scorpio,Scolopendrap and Pheretima) on adjuvant-induced arthritis rats (RA) and its mechanism of action.METHODS RA rat models were established by using Freund's adjuvant,and then the rats were divided into six groups,namely control group,model group,dexamethasone positive group,Baoguang Fengshi Liquid (Notopterygii Rhizoma et Radix,Radix angelicae pubescentis,Chuanxiong Rhizoma,etc.) positive group,and low,high doses of Hanbi Formula groups.The volume and swelling of toes were measured.The interleukin-1 β (IL-1β) and tumor necrosis factor-α (TNF-α) of serum were detected by ELISA;the proliferative capacity of lymphocytes was tested by methyl thiazolyl tetrazolium (MTI) method;synovial tissue was histopathologically examined with HE staining.Finally,the expressions of interleukin-17 (IL-17) and TNF-α in synovial tissue were determined by immunohistochemical assays.RESULTS Hanbi Formula could significantly relieve toe swelling of RA rats.Compared with the model group,Hanbi Formula could significantly alleviate synovitis in rats with RA,down-regulate the expressins of IL-1 β and TNF-α in serum and synovial tissue,and inhibit lymphocyte proliferation.There were no significant differences in above indices between low-dose and high-dose Hanbi Formula groups,which was quite with Baoguang Fengshi Liquid,but less than dexamethasone.CONCLUSION Hanbi Formula possesses an obvious function of anti-RA,and its mechanism may be related to the inhibition of lymphocyte proliferation and reducing secretion of inflammatory cytokines.
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AIM:To establish the rat model of ischemic chronic heart failure by coronary artery ligation combining with exhaustive swimming.METHODS:40 adult rats were treated with coronary artery ligation,after 4 weeks cardiac function measurement were conducted by ultrasonography.Rats with LVEF below 40％ are considered as successful model duplication.11 rats were collected for the coronary artery ligation group,while the rest (whose LVEF were bigger than 40％) were pushed to swim for 1h per day by 15 days to promote the model formation which 8 rats were collected for exhaustion with ligation group.Left ventricular function indexes,brain natriuretic peptide (BNP) and cardiac histomorphologic changing were checked,and compared with the Control group (10 rats).RESULTS:LVEF of exhaustion with ligation group was (38.70 ± 10.10) ％,coronary artery ligation group (39.20 ± 11.10)％,which was obviously decreasing (P ＜ 0.01) compared with that of the control group (84.60 ± 3.64) ％.LVEDP of exhaustion with ligation group was (11.5 ± 1.3) mm Hg,coronary artery ligation group [(10.68 ± 4.45)mm Hg],which was obviously increasing (P ＜ 0.01)compared with that of the Control group [(4.4 ± 0.2) mm Hg].The BNP level of exhaustion with ligation group was (561.0 ± 21.0) μg/L,coronary artery ligation group (548.6 ± 25.8) μg/L,which was obviously increasing (P ＜0.01) compared with that of control group [(366.2 ± 21.8) μg/L].There are lots of red myocardial cells with stripe clear in the control group based on Masson's trichrome staining,but there are so many blue collagenous fibers instead of myocardial cells in exhaustion with ligation group and coronary artery ligation group.The standard-reaching rate of model was about 35％ at 4 weeks after operation,while final standard-reaching rate rose to about 62％ after exhausting swimming.Although the difference between the indexes of the coronary artery ligation group and the post ligation group was not significant,the rate of improvement was significant (P ＜ 0.01).CONCLUSION:Ligation of coronary artery combined with swimming exhaustion can establish ischemic chronic heart failure model,which is more economic and can obtain high success rate,thus is suitable to generalization.