Efficacy of Sishen Wan(四神丸) on dinitrobenzene sulfonic acid-induced ulcerative colitis and its effect on toll-like receptor 2/interleukin-1 receptor-associated kinase-4/nuclear factor-κB signal pathway

OBJECTIVE:To investigate the therapeutic effect of Sishen Wan(四神丸,SSW)on ulcerative colitis(UC)induced by dinitrobenzene sulfonic acid and its effect on toll-like receptor 2/interleukin-1 receptor-associated kinase-4/nuclear factor-κB(TLR2/IRAK4/NF-κB)signaling pathway in colonic tissue.METHODS:In this study,120 Sprague–Dawley rats were randomly divided into blank and model groups.The experimental UC model in rats was established by subcutaneous injection of hydrocortisone+senna gavage for 21 d+dinitrobenzene sulfonic acid(DNBS)/ethanol solution enema.The successful model rats were randomly divided into the model group;mesalazine(0.36 g/kg)group;and high-,medium-,and low-dose SSW(24,12,and 6 g/kg)groups.The model and blank groups were gavaged with equal volumes of distilled water once a day for 21 d.The general condition of the rats was observed,and the body mass,fecal properties,and occult blood were recorded for calculating the disease activity index(DAI)score.The colonic tissue of the rats was collected,and its general morphology and pathological form were noted for obtaining the colonic mucosal injury index(CMDI)score.Hematoxylin-eosin staining was used to view the pathological changes of the colon tissue in each group,apoptosis of the cells was detected using terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling staining,and quantitative real-time polymerase chain reaction was used to measure the expressions of TLR2,myeloid differentiation primary response gene 88(My D88),IRAK4,and NF-κB p65 mRNA in the colon tissue.The expressions of TLR2,My D88,IRAK4,and NF-κB p65 protein were detected using western blotting and immunohistochemistry assay,and the levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in the colon tissue were determined using enzyme linked immunosorbent assay.RESULTS:Compared with the blank group,the general condition of the model group was relatively poor.The DAI and CMDI scores of the model group increased significantly(P<0.01),the glands and intestinal mucosa disappeared partially,and several inflammatory cells infiltrated and gathered in the mucosal layer and base layer of the rats in the model group.Furthermore,the cell apoptosis and expression levels of TLR2,My D88,IRAK4,and NF-κB p65 mRNA and protein in the colon tissue of rats in the model group increased significantly(P<0.01).The levels of IL-1βand TNF-αincreased significantly in the colon tissue of rats in the model group(P<0.01).After treatment with SSW,compared with the model group,the general condition of the UC rats improved.Moreover,the DAI and CMDI scores of the UC rats decreased significantly(P<0.05),and the pathological changes in the colon tissue of the UC rats tended to be normal.The cell apoptosis and expression levels of TLR2,My D88,IRAK4,and NF-κB p65 mRNA and protein in the colon tissue of the UC rats decreased gradually(P<0.01),and the levels of IL-1βand TNF-αdecreased significantly(P<0.01).CONCLUSION:SSW can improve the general condition and alleviate the intestinal mucosal injury of UC model rats.Additionally,SSW can inhibit the TLR2/IRAK4/NF-κB signaling pathway,but further studies are required to confirm it.

Angiogenesis related gene expression profiles of EA.hy926 cells induced by irbesartan： a possible novel therapeutic approach

Background Angiogenesis occurs commonly in various physiological and pathological processes. Improving blood supply through promoting angiogenesis is a novel approach for treating ischemic diseases. Angiotensin II type 1 receptor blockers （ARBs） dominate the management of hypertension, but evidence of their role in angiogenesis is contradictory. Here we explored the angiogenic effects of ARBs through characterizing gene expression of the human umbilical vein endothelial cell line EA.hy926 exposed to irbesartan. Methods The human umbilical vein endothelial cell line EA.hy926 was grown for 72 hours after treatment with different concentrations of irbesartan. The cell proliferative capacity was assessed by CCK8 assay at 24, 48 and 72 hours. Gene expression levels in EA.hy926 cells responding to irbesartan were measured under optimal proliferation conditions by microarray analysis using Affymetrix U133 plus 2.0. The differential expression of genes involved in angiogenesis was identified through cluster analysis of the resulting microarray data. Quantitative RT-PCR and Western blotting analyses were used to validate differential gene expression related to the angiogenesis process. Results In the 104, 105, 106 mol/L treatment groups, cell proliferation studies revealed significantly increased proliferation in EA.hy926 cells after 24 hours of irbesartan treatment. However, after 48 and 72 hours of treatment with different concentrations of irbesartan, there was no significant difference in cell proliferation observed in any treatment group. We selected the group stimulated with irbersartan at a concentration of 10.6 mol/L for microarray experiments. Statistical analysis of the microarray data resulted in the identification of 56 gene transcripts whose expression patterns were significantly correlated, negatively or positively, with irbesartan treatment. Cluster analysis showed that these genes were involved in angiogenesis, extracellular stimulus, binding reactions and skeletal system morphogenesis. Of these 56 genes we identified seven genes （VEGF, KDR, PTGS2, PLXND1, ROB04, LM02, and COL5A1） involved in the angiogenesis process, qRT-PCR analysis of these genes confirmed the microarray results. Protein expression of three VEGF pathway genes （VEGF, KDR, and PTGS2） was further confirmed by Western blotting. Conclusions Our study showed that irbesartan may induce angiogenic effects in vascular endothelial cells. It suggested that the mechanism of angiogenic effects of ARBs might be attributed to the signaling cascade from angiotensin receptors in the VEGF pathway. It also provided evidence indicating that ARBs could be used as a novel therapeutic approach to treat chronic ischemic heart disease as well as anti-hypertensive agents.

培哚普利联合厄贝沙坦治疗大鼠扩张型心肌病近期和远期疗效

目的:探讨培哚普利联合厄贝沙坦治疗扩张型心肌病(DCM)大鼠的近、远期疗效。方法:腹腔注射阿霉素建立大鼠DCM模型。实验大鼠分4组:A组(n=14)为正常大鼠,B组(n=26)为DCM大鼠,均无药物干预;C(n=24),D(n=24)组均为DCM大鼠,其中C组予以培哚普利2mg/(kg·d)灌胃,D组予以培哚普利1mg/(kg·d)联合厄贝沙坦25mg/(kg·d)灌胃。酶联免疫吸附法检测血浆脑利钠肽(BNP)水平;检测血钾、血肌酐水平;心肌HE染色后进行病理评分;记录干预过程各只大鼠存活期。结果:药物干预3周后,D组BNP水平低于C组(P<0.05);各组干预前后血钾、血肌酐水平差异无统计学意义(P>0.05);与B组比较,C和D组心肌病理损害均减轻(P<0.01),而C与D组间心肌病理损害的差异无统计学意义(P>0.05)。对数秩检验显示:C组存活期短于D组(P<0.05);Cox回归分析显示:联合用药或单用培哚普利为延长生存期因素,联合用药作用更明显。结论:培哚普利联合厄贝沙坦治疗DCM大鼠,在改善心功能及远期预后方面优于单用培哚普利,2种方案均能减轻心肌病理损害且未见血钾、血肌酐升高不良反应。

血管紧张素Ⅱ受体拮抗剂对糖尿病肾病蛋白尿影响的临床观察

目的观察血管紧张素Ⅱ1型受体拮抗剂缬沙坦时伴有高血压的糖尿病肾病减少蛋白尿的有效性和安全性。方法121例糖尿病肾病患者分为治疗组91例，对照组30例，两组均给予阿斯匹林100mg／d，尼群地平10mg，3次／d，治疗组在上述治疗基础上加缬沙坦160～240mg／d，疗程60mo，治疗前后行尿微量白蛋白测定。结果缬沙坦治疗组尿蛋白明显减少，自身比较及与对照组比较差异有统计学意义（P〈0．01）；缬沙坦240mg剂量优于160mg，临床转归观察治疗组完全缓解和部分缓解率为83.6％，明显高于对照组（6．7％）。结论缬沙坦可明显减少DN蛋白尿，对肾脏有明显的保护作用，并且耐受性较好。

当归注射液与血管紧张素Ⅱ受体拮抗剂对大鼠残肾模型微血管保护作用比较

目的探讨当归对大鼠残肾模型中肾脏新生血管形成的干预作用，及与缬沙坦治疗作用相比较。方法通过SD大鼠5／6肾切除建立慢性肾功能衰竭动物模型，设假手术组6只，生理盐水对照组6只，当归治疗组7只，缬沙坦治疗组6只，当归加缬沙坦治疗组6只。12周后采集血样、24h尿样及肾组织，常规病理检查判断肾小球硬化和肾小管间质损害程度；采用免疫组化染色技术，分析浸润肾组织巨噬细胞数，毛细血管密度和增生内皮细胞数。结果与假手术组相比，生理盐水对照组肾功能下降，肾间质纤维化明显，肾小球毛细血管指数（GCI）和肾小管周毛细血管指数（PCI）均显著低于假手术组（P〈0．01）。当归、缬沙坦、当归加缬沙坦治疗能显著增加肾小球和肾间质毛细血管数目（P〈0.01），并能延缓肾功能恶化、肾间质纤维化，其中GCI和PCI密度与肌酐清除率（CCr）、间质纤维化程度密切相关。并且当归不降低肾小球滤过率。与缬沙坦合用还可避免单独使用缬沙坦时CCr降低。结论当归能改善大鼠残肾模型新生血管形成和增加毛细血管密度，与缬沙坦有类似治疗作用。