The Effect of Tanshinone Ⅱ A upon the TGF-beta1/Smads Signaling Pathway in Hypertrophic Myocardium of Hypertensive Rats

SN Ⅱ A on TGF betal/Smads signal pathway in local myocardium.     

Protective Effect of Tanshinone Ⅱ A on Lipopolysaccharide-induced Lung Injury in Rats

Objective:To explore the protective effect of tanshinoneⅡA on lipopolysaccharide(LPS)-induced lung injury in rats,and possible mechanism.Methods:LPS(O111:B4) was used to produce a rat model of acute lung injury.Sprague-Dawley rats were randomly divided into 3 groups(8 in each group):the control group,the model group(ALI group),and the tanshinoneⅡA treatment group.Expression of adhesion molecule CD18 on the surface of polymorphonuclear neutrophil(PMN-CD18) in venous white blood cells(WBC),and changes in coagulation-anticoagulant indexes were measured 6 h after injection of LPS or normal saline.Changes in malondialdehyde(MDA) content,wet and dry weight(W/D) ratio and morphometry of pulmonary tissue as well as PMN sequestration in the lung were also measured.Results:(1) When compared with the control group,expression of PMN-CD18 and MDA content were enhanced in the ALI group with a hypercoagulable state(all P<0.01) and an increased W/D ratio(P<0.05).Histopathological morphometry in the lung tissue showed higher PMN sequestration,wider alveolar septa;and lower alveolar volume density(VV) and alveolar surface density(SV),showing signif icant difference(P<0.01).(2) When compared with the ALI group,the expression of PMN-CD18,MDA content,and W/D ratio were all lower in TanshinoneⅡA treatment group(P<0.05) with ameliorated coagulation abnormality(P<0.01).Histopathological morphometry in the lung tissue showed a decrease in the PMN sequestration and the width of alveolar septa(both P<0.01),and an increase in the VV and SV(P<0.05,P<0.01).Conclusion:TanⅡA plays a protective role in LPS-induced lung injury in rats through improving hypercoagulating state,decreasing PMN-CD18 expression and alleviating migration,reducing lipid peroxidation and alleviating pathological changes.     

Effect of Sodium Tanshinone ⅡA Sulfonate on Phosphorylation of Extracellular Signal-regulated Kinasel/2 in Angiotensin Ⅱ-induced Hypertrophy of Myocardial Cells

Objective： To observe the effects of sodium tanshinone ⅡA sulfonate （STS） on angiotensin Ⅱ （Ang Ⅱ）-induced hypertrophy of myocardial cells through the expression of phosphorylated extracellular signal-regulated kinase （p-ERK1/2）. Methods： In the primary culture of neonatal rat myocardial cells, the total protein content in myocardial cells was determined by coomassie brilliant blue and the protein synthesis rate was measured by [3H]-Leucine incorporation as indexes for hypertrophy of myocardial cells. The expression of p-ERK1/2 was determined using Western blot and immunofluorescence labeling. Results： （1） The total protein and protein synthesis rate increased significantly in contrast to the control group after the myocardial cells were stimulated by Ang Ⅱ （1 μ mol/L） for 24 h; STS markedly inhibited the increment of the total protein level induced by Ang Ⅱ and the syntheses of protein. （2） After pretreatment of myocardial cells with Ang Ⅱ （1 μmol/L） for 5 min, the p-ERK1/2 protein expression was increased, with the most obvious effect shown at about 10 min; pretreatment of myocardial cells with STS at different doses （2, 10, 50μmol/L） for 30 min resulted in obvious inhibition of the expression of p-ERK1/2 stimulated by Ang Ⅱ in a dose-dependent manner. （3） After the myocardial cells were stimulated by AngⅡ （1 μ mol/L）, the immunofluorescence of ERK1/2 rapidly appeared in the nucleus. The activation and translocation process of ERK1/2 induced by Ang Ⅱ was blocked distinctly by STS. （Conclusion： STS inhibited the myocardial cell hypertrophy induced by Ang Ⅱ, and the mechanism may be associated with the inhibition of p-ERK1/2 expression.     

Altered Erythrocyte Membrane Calcium Binding in Hypertensive Rats and the Effects of Sodium Tanshinone Ⅱ-A Sulphonate on It

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Effects of Tanshinone ⅡA on Transforming Growth Factor β1-Smads Signal Pathway in Renal Interstitial Fibroblasts of Rats

The effects of tanshinone ⅡA （TSN） on transforming growth factor β1 （TGFβ1） signal transduction in renal interstitial fibroblasts of rats were studied in order to investigate its mechanism in prevention of renal interstitial fibrosis. Rat renal fibroblasts of the line NRK/49F were cultured in vitro, stimulated with 5 ng/mL TGFβ1 and pretreated with 10-6, 10-5, 10-4 mol/L TSN respectively. The mRNA levels of fibronectin （FN） were examined by RT-PCR. The protein expression of FN and Smads was detected by Western blot. TGFβ1 induced the expression of FN mRNA and Smads in a time-dependent manner in a certain range. Compared with pre-stimulation, the FN mRNA and protein levels were increased by 1.1 times and 1.5 times respectively （P〈0.01, P〈0.01）, and the protein expression of phosphorylated Smad2/3 （p-Smad2/3） increased by 7 times at the end of TGFβ1 stimulation （P〈0.01）. TSN pretreatment may down-regulate the FN and p-Smad2/3 expression in a dose-dependent manner. 10-6 mol/L TSN pretreatment had no effect on the FN and p-Smad2/3 expression （both P〉0.05）. After pretreatment with 10-5 and 10-4 mol/L TSN, the FN mRNA levels were decreased by 28.1% and 43.8% respectively （P〈0.05, P〈0.01）, the FN protein levels were decreased by 40% and 44% respectively （P〈0.05, P〈0.05）, and the p-Smad2/3 protein expression were decreased by 40% and 65% respectively （P〈0.05, P〈0.01）. The inhibitory effect of TSN on renal interstitial fibrosis may be related to its blocking effect on TGFβ1-Smads signal pathway in renal intersti- tial fibroblasts.     

Effects of Tanshinone ⅡA on Transforming Growth Factor β1-Smads Signal Pathway in Renal Interstitial Fibroblasts of Rats

The effects of tanshinone ⅡA (TSN) on transforming growth factor β1 (TGFβ1) signal transduction in renal interstitial fibroblasts of rats were studied in order to investigate its mechanism in prevention of renal interstitial fibrosis. Rat renal fibroblasts of the line NRK/49F were cultured in vitro, stimulated with 5 ng/mL TGFβ1 and pretreated with 10-6, 10-5, 10-4 mol/L TSN respectively. The mRNA levels of fibronectin (FN) were examined by RT-PCR. The protein expression of FN and Smads was detected by Western blot. TGFβ1 induced the expression of FN mRNA and Smads in a time-dependent manner in a certain range. Compared with pre-stimulation, the FN mRNA and protein levels were increased by 1.1 times and 1.5 times respectively (P<0.01, P<0.01), and the protein expression of phosphorylated Smad2/3 (p-Smad2/3) increased by 7 times at the end of TGFβ1 stimulation (P<0.01). TSN pretreatment may down-regulate the FN and p-Smad2/3 expression in a dose-dependent manner. 10-6 mol/L TSN pretreatment had no effect on the FN and p-Smad2/3 expression (both P>0.05). After pretreatment with 10-5 and 10-4 mol/L TSN, the FN mRNA levels were decreased by 28.1% and 43.8% respectively (P<0.05, P<0.01), the FN protein levels were decreased by 40% and 44% respectively (P<0.05, P<0.05), and the p-Smad2/3 protein expression were decreased by 40% and 65% respectively (P<0.05, P<0.01). The inhibitory effect of TSN on renal interstitial fibrosis may be related to its blocking effect on TGFβ1-Smads signal pathway in renal intersti- tial fibroblasts.     

Protective Effect of Sodium Tanshinone ⅡA Sulfonate on Injury of Small Intestine in Rats with Sepsis and Its Mechanism

Objective:To explore the protective effect of sodium tanshinoneⅡA sulfonate(STS) on small intestine injury in rats with sepsis and its possible mechanism.Methods:According to a random number table, 24 Tats were randomly divided into 3 groups:sham operation group(sham group),sepsis model group(model group) and STS treatment group(STS group),with 8 Tats in each group.A rat model of sepsis was induced by cecal ligation and puncture(CLP) for 5 h.STS(1 mg/kg) was slowly injected through the right external jugular vein after CLP.The histopathologic changes in the intestine tissue were observed under a light microscope,and the intestinal epithelial cell apoptosis was evaluated by terminal deoxynucleoddyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL) method.The expressions of Bcl-2,Bax and nuclear factorκB(NF-κB) p65 in the intestinal tissue was determined by Western blot.The levels of tumor necrosis factorα(TNF-α) and interleukin 6(IL-6) in the intestinal tissue were determined using enzyme-linked immuno-sorbent assay(ELISA). Results:Obvious injuries were observed in the intestinal tissue in the CLP group compared with the sham group. The expression of NF-κB p65 and the levels of TNF-αand IL-6 were up-regulated after CLP,the apoptosis of intestinal epithelial cells was increased after CLP,and the ratio of Bcl-2 to Bax was decreased.STS posttreatment could attenuate the injury on the intestinal tissue induced by CLP,decrease the apoptosis of intestinal epithelial cells and the levels of NF-κB p65,TNF-αand IL-6,and increase the ratio of Bcl-2 to Bax.Conclusion: STS can protect the small intestine in rats with sepsis,and the mechanism may be associated with the inhibition of intestinal epithelial apoptosis and the reduction of activation of inflammatory cytokines.     

丹参酮IIA对COPD大鼠肺组织炎症反应的影响及相关机制研究

目的 本文主要关注丹参酮IIA对慢性阻塞性肺疾病(Chronic obstructive pulmonary disease, COPD)大鼠肺组织病理改变及肿瘤坏死因子α(Tumor Necrosis Factor α,TNFα)、白介素-1β (Interleukin-1β,IL-1β) 白介素-6(Interleukin- 6,IL-6) 及白介素-8(Interleukin- 8,IL-8)等炎症因子的影响,并探讨明确其相关机制。方法 SD大鼠被随机分为对照组、模型组、丹参酮IIA低剂量组和丹参酮IIA高剂量组。大鼠COPD模型的制备用气管内滴注脂多糖(Lipopolysaccharide, LPS)联合烟熏法。模型组大鼠每天烟熏2次,其中造模第1天、第28天每只大鼠气道内滴入脂多糖(LPS)。从造模第29天至第50天,丹参酮IIA低剂量组给予大鼠腹腔注射1 mL/( kg.d)的丹参酮ⅡA磺酸钠注射液,丹参酮IIA高剂量组给予大鼠腹腔注射2 mL/( kg.d)的丹参酮ⅡA磺酸钠注射液。对照组和模型组给予大鼠腹腔注射2mL的0.9%氯化钠溶液。于第52天收集大鼠肺组织进行病理学观察及炎性因子测定。结果 COPD模型组大鼠肺组织有明显的以淋巴细胞为主的炎性细胞浸润现象,且明显促进血清及肺泡灌洗液中炎症因子TNF-α,IL-1β,IL-6,IL-8的释放,给予丹参酮IIA高、低剂量处理组,能明显降低该炎症反应,且呈剂量依赖性。进一步研究表明,与模型组相比,丹参酮IIA高、低剂量组NF-kB p-p65的表达水平明显降低。结论 丹参酮IIA能减少COPD大鼠肺组织炎症反应,抑制炎症因子释放,是潜在的安全有效的抑制COPD的药物,且该作用可能与其抑制炎症反应的中心通路NF-kB的激活相关。     

丹参酮IIA通过抑制RIP3/FUNDC1信号通路减轻LPS诱导的小鼠肾小管上皮细胞凋亡

目的 探讨丹参酮IIA在防治脓毒血症急性肾损伤（AKI）方面的作用及潜在机制。方法 将30只C57BL/6小鼠随机分为对照组（10 mg/kg LPS等体积无菌生理盐水）、LPS组（10 mg/kg LPS作用24 h）、LPS+丹参酮IIA组（10 mg/kg 丹参酮IIA预处理15 min再给予10 mg/kg LPS作用24 h）（10只/组）。给药后检测小鼠血肌酐(Scr)、血尿素氮水平(BUN),PAS染色观察小鼠肾组织病理变化,Western blot检测小鼠肾组织RIP3、Cleaved-caspase3、p18-FUNDC1表达水平。将体外培养正常的人肾小管上皮细胞（HK-2）分为空白对照组、LPS 刺激组（LPS,10 μg/mL）、LPS+siNC 组（LPS 10 μg/mL+50 nmol/L siNC）、LPS+siRIP3 组（LPS 10 μg/mL+50 nmol/L siRIP3）、丹参酮IIA干预组（LPS 10 μg/mL+ 丹参酮IIA 10 mg/L）,分别给予以上干预措施。采用TUNEL方法检测各组HK-2细胞的凋亡情况,Western blot检测各组RIP3、Cleaved-caspase3、p18-FUNDC1表达水平,qT-PCR检测RIP3基因表达水平。结果 与对照组相比,LPS作用小鼠24 h后,小鼠Scr及血BUN水平升高,PAS染色提示近段肾小管损伤,肾组织中RIP3、Cleaved-caspase3、p18-FUNDC1蛋白表达上调（P<0.001）。与LPS组相比,丹参酮IIA预处理后,小鼠Scr及BUN水平下降,PAS染色显示近段肾小管损伤减轻,肾组织中RIP3、Cleaved-caspase3、p18- FUNDC1蛋白表达下降（P<0.001）。体外研究显示,与对照组相比,LPS刺激的HK-2细胞后,TUNEL染色显示细胞凋亡水平明显增加,Cleaved-caspase3、RIP3、p18-FUNDC1表达上调（P<0.05）。应用丹参酮IIA预处理或体外沉默RIP3表达后再次予以LPS刺激细胞,TUNEL染色显示细胞凋亡水平较LPS组明显减少,Cleaved-caspase3、RIP3、p18-FUNDC1表达水平较LPS组下降（P<0.05）。结论 丹参酮IIA可能通过抑制RIP3/ FUNDC1信号通路来改善LPS诱导的肾小管上皮细胞凋亡。     

丹参酮IIA对跟腱损伤模型大鼠肌腱粘连的预防作用研究

目的 研究丹参酮IIA对肌腱粘连的预防作用和对肌腱愈合的影响。方法 将20只跟腱损伤大鼠随机分为实验组和对照组各10只,其中实验组局部注射丹参酮IIA注射液0.2ml,对照组注射等量生理盐水。通过大体解剖观察及组织学评价比较两组的肌腱粘连和愈合情况。结果 两组大鼠肌腱均顺利愈合。肌腱粘连评分显示实验组较对照组肌腱粘连少,程度轻,两者具有统计学差异[（2.9±0.7）分比（4.0±1.6）分,P＜0.05]。组织学观察显示实验组肌腱损伤处炎性细胞浸润少,胶原纤维和成纤维细胞增生比对照组少,排列较规则。结论 丹参酮IIA可以减少肌腱损伤术后的粘连,且对肌腱愈合无负面影响。     

灯盏花素对糖尿病大鼠心肌转化生长因子-β1和Smad7表达的影响

目的 探讨灯盏花素对糖尿病大鼠心肌转化生长因子-β1(TGF-β1)和Smad7表达的影响.方法 健康SD大鼠30只,随机选取20只造模成功后分为糖尿病组(DM组)和灯盏花素治疗组(Bre组)各10只,余10只作为正常对照组(NC组).Bre组给予灯盏花素注射液20 mg/(kg*d)腹腔内注射,DM组和NC组等量生理盐水腹腔内注射,12周后,尾静脉测血糖,测定大鼠心功能,计算心指数,光镜观察心肌结构病理改变,电镜观察心肌超微结构改变,免疫组化法检测大鼠心肌TGF-β1、Smad7蛋白表达水平.结果 Bre组与DM组间血糖值差异无统计学意义(P>0.05);灯盏花素能显著降低糖尿病大鼠的心指数,明显升高左心室内压最大上升和下降速率,显著减轻心肌病理改变,减少心肌TGF-β1和增加Smad7的表达(P<0.05,P<0.01).结论 灯盏花素可减轻糖尿病大鼠心肌的病理改变,保护糖尿病大鼠心脏功能,其机制可能是通过改善心肌中TGF-β1、Smad7的表达,从而抑制糖尿病大鼠心肌的纤维化来发挥对糖尿病大鼠心脏的保护作用.     

氯沙坦通过AT1R/VEGF 信号通路对肝癌血管生成的影响

目的 探讨氯沙坦是否通过下调血管紧张素Ⅱ 1 型受体（AT1R）/ 血管内皮生长因子（VEGF）信号通路,抑制肝癌细胞的血管生成。方法 采用免疫荧光染色和Western blot 检测AT1R 在肝癌细胞系HepG2、HuH-7 和PLC/PRF/5,以及正常肝细胞LO2 中的表达。采用Western blot 检测氯沙坦对肝癌细胞系HepG2 中AT1R 表达的影响,酶联免疫吸附法（ELISA）检测氯沙坦对肝癌细胞系HepG2 中VEGF 的影响。采用免疫组织化学法检测氯沙坦对大鼠肝癌组织中AT1R、VEGF 和CD34 表达的影响。结果 AT1R 在正常肝细胞LO2 中低表达（P <0.05）,在肝癌细胞系HepG2、HuH-7 和PLC/PRF/5 中高表达（P <0.05）,在HepG2 细胞中表达最高（P <0.05）。在肝癌细胞系HepG2 中加入氯沙坦后,AT1R 蛋白水平和VEGF 浓度降低（P <0.05）。大鼠肝癌组织中AT1R、VEGF 和CD34 高表达,而加入氯沙坦后,AT1R、VEGF 和CD34 表达降低（P <0.05）。结论 氯沙坦通过抑制AT1R/VEGF 信号通路,可以阻碍肝癌血管的生成。     

滋阴补阳序贯法联合西药对卵巢储备功能下降排卵障碍性不孕大鼠TGF-β1/Smads信号通路的影响

目的：探讨滋阴补阳序贯法联合西药对卵巢储备功能下降(diminished ovarian reserve,DOR)排卵障碍性不 孕大鼠的TGF-β1/Smads信号通路的影响。方法：SD雌性大鼠40只,随机分为空白组、模型组、西药组(人工周期+促 排卵)、滋阴补阳序贯组、联合用药组共5组,每组8只。用雷公藤多苷片50 mg/(kg.d)灌胃连续2周,建立DOR大鼠模 型。空白组、模型组用生理盐水灌胃,连续10日;西药组予人工周期加促排卵治疗;滋阴补阳序贯组在动情前期予 滋阴方,动情后期予补阳方灌胃;联合用药组在西药组的基础上动情前期予滋阴方,动情后期予补阳方。HE染色 观察大鼠卵巢组织形态变化,免疫组织化学检测大鼠卵巢转化生长因子β1受体(transforming growth factor β1 receptor, TGF-β1R)的水平,Western印迹检测大鼠卵巢Smad2,Smad3,Smad7蛋白的表达。结果：与空白组比较,DOR模型组 大鼠卵巢黄体数、成熟卵泡数、生长卵泡数显著减少,闭锁卵泡数升高(P<0.01);TGF-β1R表达升高,Smad2和Smad3 蛋白水平显著降低,Smad7蛋白显著升高(P<0.01)。与模型组比较,各治疗组黄体数、成熟卵泡数、生长卵泡数增 多,闭锁卵泡数减少,Smad2和Smad3蛋白水平升高,Smad7蛋白水平降低(P<0.05或P<0.01)。联合用药组与西药组 比较,黄体数及生长卵泡数增加,TGF-β1R表达升高,Smad2和Smad3蛋白水平升高,Smad7蛋白水平降低(P<0.05或 P<0.01)。结论：滋阴补阳序贯法联合西药能够上调DOR大鼠卵巢TGF-β1R,Smad2,Smad3表达,降低Smad7表达,改 善DOR大鼠卵巢储备功能。     

螺内酯和缬沙坦对高血压大鼠心肌肥大信号通路的影响

目的观察血管紧张素Ⅱ受体拮抗剂缬沙坦和盐皮质激素受体拮抗剂螺内酯对自发性高血压大鼠(spontane-ously hypertensive rats,SHR)心肌中活化的丝裂原活化蛋白激酶家族(m itogen-activated protein kinase fam ily,MAPK)(细胞外信号调节激酶[ERK]、c-Jun NH2-末端激酶[JNK]和p38)的影响。方法将18只雄性SHR随机分为3组,每组6只。其中两组分别用缬沙坦30 mg.kg-1.d-1、螺内酯20 mg.kg-1.d-1溶于饮水灌胃,连续治疗13周;对照组给正常饮水,并与W istar-kyoto大鼠(WKY)比较。用W estern-blot方法检测大鼠心肌磷酸化MAPK的表达。结果SHR对照组心肌磷酸化JNK/actin值高于其余3组(P<0.01),缬沙坦组高于螺内酯组和WKY组(P<0.01),螺内酯组与WKY水平接近;SHR对照组心肌磷酸化ERK/actin值高于其余3组(P<0.01),螺内酯和缬沙坦组高于WKY组(P<0.01),两用药组之间无差异;缬沙坦组磷酸化p38/actin值高于WKY组(P<0.01),低于SHR对照组和螺内酯组(P<0.01)。结论醛固酮能促进SHR的心肌肥厚和心肌纤维化,盐皮质激素受体拮抗剂能通过抑制MAPK途径而抑制左室肥厚和心肌纤维化。     

Effect of Sodium Tanshinone Ⅱ A Sulfonate on Phosphorylation of Extracellular Signal-regulated Kinase1/2 in Angiotensin Ⅱ-induced Hypertrophy of Myocardial Cells

Objective:To observe the effects of sodium tanshinone Ⅱ A sulfonate(STS)on angiotensin Ⅱ(Ang Ⅱ)-induced hypertrophy of myocardial cells through the expression of phosphorylated extracellular signal-regulated kinase(P-ERK1/2).Methods:In the primary culture of neonatal rat myocardial cells.the total protein content in myocardial cells was determined by coomassie brilliant blue and the protein synthesis rate was measured by[3H]-Leucine incorporation as indexes for hypertrophy of myocardial cells.The expression of p-ERK1/2 was determined using Western blot and immunofluorescence Iabeling.Results:(1)The totaI protein and protein synthesis rate increased significantly in contrast to the control group after the myocardial cells were stimulated by Ang Ⅱ (1 μmol/L)for 24 h;STS markedly inhibited the increment of the total protein level induced by Ang Ⅱ and the syntheses of protein.(2)After pretreatment of myocardial cells with Ang Ⅱ(1 μ mol/L)for 5 min,the p-ERK1/2 protein expression was increased,with the most obvious effect shown at about 10 min;pretreatment of myocardial cells with STS at different doses(2,10,50 μ mol/L)for 30 min resulted in obvious inhibition of the expression of p-ERK1/2 stimulated by Ang Ⅱ in a dose-dependent manner.(3)After the myocardial cells were stimulated by Ang Ⅱ(1 μ mol/L),the immunofluorescence of ERK1/2 rapidly appeared in the nucleus.The activation and translocation process of ERK1/2 induced by Ang Ⅱ was blocked distinctly by STS.Conclusion:STS inhibited the myocardial cell hypertrophy induced by Ang Ⅱ,and the mechanism may be associated with the inhibition of p-ERK1/2 expression.