丹参酮生物合成相关SmERF108转录因子的鉴定及其靶基因分析＊

目的 鉴定与丹参酮合成相关转录因子AP2/ERF家族中SmERF108转录因子，并分析转录因子SmERF108的靶基因。方法 利用生物信息学在线分析平台如NCBI、PFAM等分析SmERF108的序列特征；MEGA-X软件用于构建SmERF108与不同功能转录因子的系统进化树；拟南芥原生质体转化法鉴定SmERF108蛋白的亚细胞定位；通过实时荧光定量PCR（Real-time qPCR）对SmERF108基因在丹参不同器官和组织差异表达进行检测；利用酵母体系对SmERF108的转录激活活性进行探究并用酵母单杂技术确定其靶基因。结果 SmERF108具有典型的AP2/ERF保守结构域，属于ERF-B3亚组，系统进化树和保守基序分析显示SmERF108与丹参中SmERF128、青蒿中AaERF2亲缘关系较近且保守基序分布一致；亚细胞定位显示SmERF108蛋白定位于细胞核；SmERF108基因在周皮中表达最高，且呈现周皮（R1）>韧皮部（R2）>木质部（R3）的规律；酵母自激活验证SmERF108具有转录激活活性，同时酵母单杂交确认其能与关键酶基因SmCPS1启动子结合。结论 鉴定到丹参中一个新转录因子SmERF108，生物信息学分析及差异表达分析预测与丹参酮合成相关，分子互作初步证实靶基因为SmCPS1二萜环化酶。     

丹参酮ⅡA对压力超负荷大鼠心肌骨桥蛋白表达的影响

目的:观察丹参酮ⅡA(TanⅡA)对压力超负荷大鼠心肌骨桥蛋白表达的影响,探讨TanⅡA防治心肌纤维化的作用机制。方法:在50只雄性6周龄SD大鼠中随机取42只行腹主动脉缩窄术制备压力超负荷模型(手术组),另外8只大鼠行假手术(假手术组)。4周后,手术组大鼠死亡12只,假手术组大鼠无死亡。将存活的30只手术组大鼠随机分为模型组、TanⅡA高剂量组和TanⅡA低剂量组。给药4周后,处死所有大鼠,HE和Masson染色观察大鼠心肌病理学改变,测量胶原容积分数(CVF)和血管周围胶原容积分数(PCVF)。碱水法检测心肌羟脯氨酸含量。免疫组织化学法分析心肌α-平滑肌肌动蛋白(α-SMA)、结缔组织生长因子(CTGF)和骨桥蛋白的表达水平。结果:TanⅡA能显著减少压力超负荷大鼠心肌CVF、PCVF、羟脯氨酸含量,降低α-SMA、CTGF、骨桥蛋白的表达水平(P0.05),且高剂量TanⅡA的改善作用更为明显。结论:TanⅡA改善压力超负荷大鼠心肌纤维化的作用机制可能与抑制骨桥蛋白的表达有关。     

丹参酮ⅡA诱导瘢痕疙瘩成纤维细胞凋亡及细胞周期阻滞的实验研究

目的观察丹参酮ⅡA对人瘢痕疙瘩成纤维细胞增殖、凋亡及细胞周期的影响。方法体外培养人瘢痕疙瘩成纤维细胞,予含有不同浓度丹参酮ⅡA的培养液(分别为0μg/mL、50μg/mL、100μg/mL和200μg/mL)进行干预。不同时间点以CCK-8检测细胞增殖活力,流式细胞仪检测细胞凋亡、细胞周期的变化。结果不同浓度丹参酮ⅡA干预和不同时间干预,对瘢痕疙瘩成纤维细胞的增殖活性、早期凋亡率、细胞周期G0/G1期比例的影响,均具有统计学意义(P<0.001),其中200μg/mL组干预效果最明显。结论丹参酮ⅡA具有抑制瘢痕疙瘩成纤维细胞增殖,诱导凋亡并阻滞细胞周期的作用,且干预效果存在浓度依耐性及时间依耐性。     

丹参酮ⅡA逆转高糖诱导一氧化氮生成减少及机制研究

目的：观察丹参酮ⅡA对高糖引起内皮细胞中一氧化氮生成量减少的影响,并探讨相关的作用机制。方法：将内皮细胞随机分为空白对照组,等渗对照组,高糖处理组,溶剂对照组,丹参酮ⅡA组。一氧化氮特异性探针检测法检测内皮细胞一氧化氮含量。分别采用酶联免疫吸附(ELISA)和Griess法检测环磷酸鸟苷(cGMP)和总硝基化合物的含量。免疫印迹法和实时荧光定量聚合酶链反应(qPCR)分别检测内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)蛋白和mRNA水平的变化。结果：丹参酮ⅡA可抑制高糖诱导的人脐静脉内皮细胞中一氧化氮生成量(P<0.05), cGMP含量以及总硝基化合物生成的减少(P<0.05),并能逆转eNOS蛋白表达的降低(P<0.05)。结论：丹参酮ⅡA通过上调eNOS蛋白表达抑制一氧化氮生成的减少。     

Tanshinone IIA attenuates interleukin-17A-induced systemic sclerosis patient-derived dermal vascular smooth muscle cell activation via inhibition of the extracellular signal-regulated kinase signaling pathway

OBJECTIVE:Salvia miltiorrhiza has long been used to treat systemic sclerosis. Tanshinone IIA, one of the phytochemicals derived from the roots of Salvia miltiorrhiza, exhibits multiple biological activities. The present study aimed to investigate whether tanshinone IIA has an effect on the interleukin-17A-induced functional activation of systemic sclerosis patient-derived dermal vascular smooth muscle cells.METHODS:Systemic sclerosis patient-derived dermal vascular smooth muscle cells were incubated with various dosages of tanshinone IIA in the presence of interleukin-17A or the serum of systemic sclerosis patients. Cell proliferation was assessed using Cell Counting Kit-8. The expression of collagen 1 and 3 in cells was evaluated by immunofluorescence. Cell migration was measured using a transwell assay. The expression of phospho-extracellular signal-regulated kinase was detected by Western blotting.RESULTS:Our data demonstrate that tanshinone IIA exerts an inhibitory effect on interleukin-17A-induced systemic sclerosis patient-derived dermal vascular smooth muscle cell proliferation, collagen synthesis and migration.CONCLUSION:These findings suggest that tanshinone IIA might serve as a promising therapeutic agent for the treatment of systemic sclerosis.     

丹参酮ⅡA通过调节Th1/Th2平衡改善大鼠自身免疫性心肌炎的机制研究

目的：探讨丹参酮ⅡA通过调节Th1/Th2平衡改善大鼠自身免疫性心肌炎(experimental autoimmune myocarditis,EAM)的机制。方法：选取6周龄Lewis大鼠，随机分为对照组、EAM造模组（A组）、EAM造模+低剂量丹参酮IIA治疗组（B组）、EAM造模+高剂量丹参酮IIA治疗组（C组）。初次免疫后21天，超声心动图检测大鼠心功能；处死大鼠，取静脉血用于血清IL-2、IFN-γ、IL-4、IL-10检测；取心脏称重，计算心脏重量/体重比值，并行组织病理学检查；取脾脏，流式分析脾脏Th细胞分化情况。结果：（1）组织形态学结果：A组大鼠心脏重量/体重比值、病理学评分较对照组显著升高，B、C组大鼠较A组有不同程度下降（P<0.05）；（2）超声心动图检测结果：A组大鼠左室收缩末内径（LVEDs）、左室舒张末内径（LVEDd）、室间隔厚度（IVS）、左室后壁厚度（LVPW）较对照组显著增加，左室短轴缩短率（LVFS）显著降低（P<0.05），C组大鼠LVEDs、LVEDd、IVS、LVPW均较A组显著降低，LVFS显著增加（P<0.05）；（3）脾脏Th细胞分化情况：A组大鼠脾脏Th1细胞比例及Th1/Th2比值均较对照组显著升高（P<0.05）；给予丹参酮ⅡA治疗后，Th1细胞比例及Th1/Th2比值较A组显著降低，Th2细胞比例显著升高（P<0.05），且呈现剂量依赖性；（4）血清Th1/Th2细胞因子检测：A组大鼠血清Th1型细胞因子（IL-2、IFN-γ）水平较对照组显著升高，Th2型细胞因子（IL-4、IL-10）水平显著降低（P<0.05）；给予丹参酮ⅡA治疗，B、C组大鼠血清IL-2、IFN-γ水平较A组显著降低， IL-4、IL-10水平显著升高（P<0.05），且呈现剂量依赖性。结论：丹参酮ⅡA可通过上调Th2细胞比例，维持Th1/Th2细胞平衡，从而改善大鼠自身免疫性心肌炎所致的心肌损伤，恢复心脏功能。     

Tanshinone IIA protects rat primary hepatocytes against carbon tetrachloride toxicity via inhibiting mitochondria permeability transition

Tanshinone IIA (Tan IIA), one of the key components of Salvia milthorrhiza Bunge (Lamiaceae), is used to treat liver disease. The present study was carried out to investigate the possible mechanisms involved in the hepatoprotective effects of Tan IIA on carbon tetrachloride (CCl₄)-induced hepatocyte toxicity. In cultures treated with 1 or 2 μM CCl₄, Tan IIA (10–75 μM) significantly increased hepatocyte survival rates. However, only at a concentration of 75 μM could Tan IIA partially reverse the CCl₄ (3 μM)-induced decrease of survival rate (34?±?3% vs. 18?±?3%, n?=?8, p?<?0.01). In isolated mitochondria energized with succinate, Tan IIA could inhibit the large swelling effect induced by CCl₄ (1 and 2 μM). Base on these results, Tan IIA could protect rat primary cultured hepatocytes from CCl₄-induced toxicity partially by the inhibitory effect on the opening of mitochondrial permeability transition (MPT).     

成纤维细胞生长因子2和丹参酮ⅡA在大鼠骨髓间充质干细胞向心肌样细胞分化中的作用

目的 探讨成纤维细胞生长因子2（FGF-2）和丹参酮ⅡA（tanshinoneⅡA）在大鼠骨髓间充质干细胞（BMSCs）向心肌样细胞分化中的作用。 方法 体外分离、培养BMSCs,利用流式细胞术进行鉴定。实验分组：对照组（不加任何诱导剂）、FGF-2组、丹参酮ⅡA组及两者联合诱导组。MTT检测诱导后的活性及增殖情况;Real-time PCR检测早期心肌转录因子GATA-4、Nkx2.5的表达;免疫细胞化学染色法检测缝隙连接蛋白43(Cx43)以及心肌肌钙蛋白Ⅰ（cTnI）的表达;免疫荧光化学染色法检测结蛋白(desmin)、原肌球蛋白（Tm）的表达;Western blotting检测结蛋白、Tm的表达。 结果 各诱导组较对照组增殖明显。与对照组相比,诱导组GATA-4和Nkx2.5基因的表达增强,联合组表达量最高,差异具有统计学意义（P＜0.05）。联合诱导组各标记物Cx43、cTnI、结蛋白、Tm的阳性表达率高于FGF-2及丹参酮ⅡA单独诱导组,差异具有统计学意义（P＜0.05）。Western blotting结果显示,联合诱导组结蛋白、Tm的表达量明显高于其他实验组,差异具有统计学意义（P＜0.05）。透射电子显微镜结果显示,诱导后细胞的细胞核居中,细胞质中可见肌丝、粗面内质网、线粒体和核糖体。 结论 FGF-2和丹参酮ⅡA均能促进BMSCs增殖,诱导BMSCs分化为心肌样细胞,两者联合诱导的效果较其他组更佳。     

HPLC法测定加味逍遥散复方中丹参酮ⅡA的含量

目的 建立加味逍遥散复方中丹参酮ⅡA的测定方法。方法 采用十八烷基硅烷键合硅胶柱（inertsil ODS-3柱 250 mm×4.6 mm,5 μm）;流动相:乙腈-0.1%磷酸,进行梯度洗脱;检测波长:270 nm;流速:1.0 ml/min;柱温:20℃。结果 丹参酮ⅡA在0.0001~0.0005 μg范围内呈现良好的线性关系[r=0.9991,平均回收率为97.36%（RSD=1.38%,n=6）]。结论 本方法专属性强,准确度高,重现性良好,可用于加味逍遥散的质量控制。     

Tanshinone IIA Ameliorates Trinitrobenzene Sulfonic Acid (TNBS)-Induced Murine Colitis

Inflammatory bowel diseases are characterized by proinflammatory cytokines, oxidative stress, and tissue damage. Recently, tanshinone had been shown to act as an antioxidant, and to have anti-inflammatory bioactivity. The study was carried out to investigate the effect of tanshinone IIA on the inflammatory response of experimental colitis. Murine colitis was induced by trinitrobenzene sulfonic acid (TNBS). Ten or 20 mg tanshinone IIA was administrated to mice 4 h before the induction of colitis, and repeated daily until the mice were sacrificed. Colonic inflammation was examined by histological analysis, myeloperoxidase (MPO) activity, and the production of proinflammatory cytokines in colonic tissue. Activation of nuclear factor-kappa B was identified by western blot and immunohistochemistry, and oxidative stress was shown by glutathione (GSH) level in tissue. The mice with colitis treated by tanshinone IIA showed less tissue damage, lower MPO activity, less production of TNF-α and IL-1β, a higher level of GSH in colonic tissue, and downregulated activation of nuclear factor-kappa B in lamina propria mononuclear cells, compared with those of the untreated colitis group. Our data indicates that tanshinone IIA inhibits inflammatory response of colitis by downregulating the production of proinflammatory cytokines, and attenuating oxidative stress, which suggests that tanshinone IIA may be a new potential management for inflammatory bowel diseases.