丹参酮ⅡA磺酸钠联合卡维地洛对心力衰竭患者心功能及内分泌激素水平的影响

目的探讨丹参酮Ⅱ_A磺酸钠联合卡维地洛对心力衰竭患者心功能及内分泌激素水平的影响。方法选取医院2017年8月至2019年8月收治的心力衰竭患者96例,按随机数字表法分为观察组和对照组,各48例。两组均予卡维地洛治疗,观察组加用丹参酮Ⅱ_A磺酸钠治疗。两组均连续治疗2周。结果观察组总有效率为89.58%,显著高于对照组的72.92%(P <0.05);治疗后,观察组患者左室射血分数(LVEF)显著高于对照组,左室收缩末期内径(LVESd)、左室舒张末期内径(LVEDd)均显著低于对照组(P <0.05),内分泌激素[血管紧张素Ⅱ(Ang-Ⅱ)、醛固酮(ALD)、血管内皮素(ET)]水平均显著低于对照组(P <0.05),炎性因子[C反应蛋白(CRP)、肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)]水平均显著低于对照组(P <0.05);治疗期间,观察组与对照组患者不良反应发生率相当(18.75%比10.42%,P> 0.05)。结论丹参酮Ⅱ_A磺酸钠联合卡维地洛治疗心力衰竭临床疗效显著,能改善患者心功能,降低内分泌激素和炎性因子水平,且安全性良好。     

丹参酮II-A通过调控TLR4/IκBα/NFκB信号通路抑制LPS诱导的细胞炎症

目的建立脂多糖(LPS)诱导的小鼠单核巨噬细胞(RAW264.7)炎症模型,探究丹参酮II-A(Tan IIA)的抗炎活性及其机制。方法CCK-8法测定Tan IIA对细胞活力的影响;迁移小室测定Tan IIA对LPS诱导细胞迁移能力作用;ELISA法测定细胞上清液中小鼠肿瘤坏死因子α(tumor necrosis factoralpha,TNF-α)、白介素6(interleukin 6,IL-6)、IL^-1β、单核细胞趋化蛋白-1(monocyte chemoattractant protein,MCP-1)的含量;Western blot法检测基质金属蛋白酶2(matrix metalloproteinases,MMP-2)、MMP-9、Toll样受体-4(TLR4)、IκB-α、p-IκB-α、NFκB和p-NFκB蛋白的表达。结果Tan IIA对LPS诱导的RAW264.7细胞培养液中炎症因子TNF-α、IL-6、IL^-1β和MCP-1的分泌有明显的抑制作用;明显下调MMP-2、MMP-9、TLR4、p-IκB-α和p-NFκB的蛋白的表达,抑制IκB-α磷酸化和NFκB的入核和活化。结论Tan IIA可通过抑制MMP-2和MMP-9的表达以及TLR4/κB-α/NF-κB信号通路,调控TNF-α、IL-6、IL^-1β等炎症因子的释放而发挥抗炎活性。     

基于HPLC-QAMS法的清肝降压胶囊中10个成分同时定量测定研究

目的:建立同时测定清肝降压胶囊中10个成分的高效液相色谱一测多评(HPLC-QAMS)方法,并验证其准确性和可行性。方法:采用Phenomsil C18色谱柱(4.6 mm×250 mm,5μm),以乙腈-0.1%磷酸水溶液为流动相,梯度洗脱;流速:1.0 mL·min^-1;柱温:30℃;检测波长:320 nm(检测2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷)、250 nm(检测3'-羟基葛根素、葛根素和3'-甲氧基葛根素)、208 nm(检测泽泻醇F、泽泻醇A、24-乙酰泽泻醇A和23-乙酰泽泻醇B)和270 nm(检测隐丹参酮和丹参酮ⅡA)。以葛根素为内参物,建立其与其他9种成分的相对校正因子(RCF),并计算含量,实现一测多评;同时采用外标法(ESM)测定清肝降压胶囊中10个成分的含量,比较一测多评法计算值与外标法实测值的差异。结果:10个成分的线性范围分别为8.136~162.7、7.488~149.8、30.54~610.9、14.11~282.2、0.3680~7.360、0.7040~14.08、0.4320~8.640、3.056~61.12、0.5120~10.24、0.8480~16.96μg·mL^-1,r为0.9992~0.9999;平均回收率分别为98.5%、98.0%、100.0%、100.0%、97.8%、98.5%、97.0%、99.1%、99.3%和100.1%,RSD为1.3%、1.1%、0.96%、0.64%、1.6%、1.5%、1.2%、0.83%、1.1%和0.72%;葛根素与2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、3'-羟基葛根素、3'-甲氧基葛根素、泽泻醇F、泽泻醇A、24-乙酰泽泻醇A、23-乙酰泽泻醇B、隐丹参酮、丹参酮ⅡA的相对校正因子分别为1.1551、1.1262、1.2760、0.9643、0.8959、0.8593、1.3662、0.8399和0.9663;一测多评法计算值和外标法实测值之间无明显差异。结论:所建立的方法简便、准确,可用于清肝降压胶囊的质量评价研究。     

Tanshinone IIA prevents LPS-induced inflammatory responses in mice via inactivation of succinate dehydrogenase in macrophages

Metabolic reprogramming is associated with NLRP3 inflammasome activation in activated macrophages, contributing to inflammatory responses. Tanshinone IIA (Tan-IIA) is a major constituent from Salvia miltiorrhiza Bunge, which exhibits anti-inflammatory activity. In this study, we investigated the effects of Tan-IIA on inflammation in macrophages in focus on its regulation of metabolism and redox state. In lipopolysaccharides (LPS)-stimulated mouse bone marrow-derived macrophages (BMDMs), Tan-IIA (10 μM) significantly decreased succinate-boosted IL-1β and IL-6 production, accompanied by upregulation of IL-1RA and IL-10 release via inhibiting succinate dehydrogenase (SDH). Tan-IIA concentration dependently inhibited SDH activity with an estimated IC₅₀ of 4.47 μM in LPS-activated BMDMs. Tan-IIA decreased succinate accumulation, suppressed mitochondrial reactive oxygen species production, thus preventing hypoxia-inducible factor-1α (HIF-1α) induction. Consequently, Tan-IIA reduced glycolysis and protected the activity of Sirtuin2 (Sirt2), an NAD⁺-dependent protein deacetylase, by raising the ratio of NAD⁺/NADH in activated macrophages. The acetylation of α-tubulin was required for the assembly of NLRP3 inflammasome; Tan-IIA increased the binding of Sirt2 to α-tubulin, and thus reduced the acetylation of α-tubulin, thus impairing this process. Sirt2 knockdown or application of Sirt2 inhibitor AGK-2 (10 μM) neutralized the effects of Tan-IIA, suggesting that Tan-IIA inactivated NLRP3 inflammasome in a manner dependent on Sirt2 regulation. The anti-inflammatory effects of Tan-IIA were observed in mice subjected to LPS challenge: pre-administration of Tan-IIA (20 mg/kg, ip) significantly attenuated LPS-induced acute inflammatory responses, characterized by elevated IL-1β but reduced IL-10 levels in serum. The peritoneal macrophages isolated from the mice displayed similar metabolic regulation. In conclusion, Tan-IIA reduces HIF-1α induction via SDH inactivation, and preserves Sirt2 activity via downregulation of glycolysis, contributing to suppression of NLRP3 inflammasome activation. This study provides a new insight into the anti-inflammatory action of Tan-IIA from the respect of metabolic and redox regulation.     

丹参酮ⅡA对过敏性紫癜肾炎小鼠肾组织中Cosmc和AOPP表达的影响及其治疗作用

目的：探讨丹参酮ⅡA对过敏性紫癜肾炎小鼠肾组织中半乳糖基转移酶伴侣蛋白（Cosmc）、晚期氧化蛋白终末产物（AOPP）表达及胞外信号调节激酶/丝裂原活化蛋白激酶（ERK/MAPK）信号通路蛋白水平的影响,阐明丹参酮ⅡA对过敏性紫癜肾炎的治疗机制。方法：38只小鼠随机分为对照组、模型组（建立过敏性紫癜肾炎模型）和丹参酮ⅡA组（建立过敏性紫癜肾炎模型+丹参酮ⅡA治疗）,其中2只小鼠用于验证造模是否成功,其余每组12只。测定各组小鼠尿红细胞计数和尿蛋白水平,采用过碘酸希夫反应（PAS）染色观察各组小鼠肾组织病理形态表现,采用酶联免疫吸附法（ELISA）测定小鼠肾组织中AOPP蛋白水平,采用Western blotting法测定小鼠肾组织中ERK和磷酸化ERK（p-ERK）蛋白表达水平。采用逆转录-聚合酶链反应（RT-PCR）法测定各组小鼠肾组织中ERK、p-ERK和Cosmc mRNA表达水平。结果：对照组小鼠肾小球基底膜无增生,无肾小球硬化;模型组小鼠肾小球基底膜增生明显,可见炎症细胞浸润,出现肾小球硬化;丹参酮ⅡA组小鼠肾小球基底膜增生和炎症浸润不明显,少量炎症细胞浸润。与对照组比较,模型组和丹参酮ⅡA组小鼠尿红细胞计数、24 h尿蛋白水平及肾组织中AOPP蛋白水平和p-ERK mRNA表达水平升高（P<0.05或P<0.01）,Cosmc mRNA表达水平降低（P<0.01）;与模型组比较,丹参酮ⅡA组小鼠尿红细胞计数、24 h尿蛋白水平及肾组织中AOPP蛋白水平和p-ERK蛋白表达水平降低（P<0.05或P<0.01）,Cosmc mRNA表达水平升高（P<0.01）。模型组小鼠肾组织中Cosmc mRNA表达水平与AOPP蛋白水平及p-ERK蛋白表达水平呈负相关关系（r=-0.573,P<0.01;r=-0.602,P<0.01）,AOPP蛋白水平与p-ERK蛋白表达水平呈正相关关系（r=0.614,P<0.01）。结论：丹参酮ⅡA可能通过降低肾组织Cosmc水平、升高肾组织中AOPP水平以及激活ERK/MAPK信号通路对过敏性紫癜肾炎小鼠发挥治疗作用。     

丹参酮ⅡA对人胎盘间充质干细胞向心肌细胞分化的诱导作用及其机制

目的：探讨丹参酮ⅡA （TanⅡA）对人胎盘间充质干细胞（hPDMSCs）向心肌细胞分化的诱导作用及其机制,为TanⅡA作为心肌细胞分化诱导剂提供实验依据。方法：采用不同浓度TanⅡA （0.1、0.2、0.4、0.6、0.8、1.0、2.0、4.0、6.0、8.0和10.0 mg·L^-1）处理hPDMSCs,MTT法筛选出无毒剂量的TanⅡA （0.1 mg·L^-1）用于实验。hPDMSCs培养体系分为对照组、5-氮胞苷（5-aza,10μmol·L^-1）诱导组和TanⅡA （0.1 mg·L^-1）诱导组。培养20d后,免疫组织化学法检测各组细胞中α-横纹肌肌动蛋白（α-SCA）的表达;免疫荧光法检测各组细胞中心肌肌钙蛋白I （cTnI）的阳性表达率,计算心肌细胞分化率;Western-blotting法检测各组细胞中心肌转录调节因子4（GATA4）、心钠素（ANF）、cTnI、糖原合成酶激酶-3β（GSK-3β）和β-连环蛋白（β-catenin）的蛋白表达水平。结果：hPDMSCs的生物学特性符合间充质干细胞。MTT法,当TanⅡA浓度大于0.1 mg·L^-1时,细胞存活率随浓度的增加而降低。对照组细胞在培养12 d以前呈快速增长趋势,培养12 d后细胞增殖活性降低;与对照组比较,5-aza诱导组和TanⅡA诱导组细胞增殖活性明显降低（P<0.05）。免疫组织化学染色,对照组细胞不表达α-SCA,5-aza诱导组和TanⅡA诱导组细胞均表达α-SCA,且TanⅡA诱导组更明显。与对照组比较,5-aza诱导组和TanⅡA诱导组细胞中GATA4（t_5-aza=2.937,P_5-aza<0.05;t_TanⅡA=4.769,P_TanⅡA<0.05）、ANF （t_5-aza=3.728,P_5-aza<0.05;t_TanⅡA=5.912,P_TanⅡA<0.05）、cTnI （t_5-aza=3.623,P_5-aza<0.05;t_TanⅡA=7.153,P_TanⅡA<0.05）和GSK-3β（t_5-aza=2.995,P_5-aza<0.05;t_TanⅡA=5.420,P_TanⅡA<0.05）蛋白表达水平明显升高,β-catenin （t_5-aza=2.985,P_5-aza<0.05;t_TanⅡA=6.951,P_TanⅡA<0.05）蛋白表达水平明显降低;与5-aza诱导组比较,TanⅡA诱导组GATA4、ANF和GSK-3β蛋白表达水平进一步升高（P<0.05）。结论：TanⅡA能诱导hPDMSCs分化为心肌细胞,且效果优于5-aza,其机制可能与TanⅡA能抑制Wnt/β-catenin信号通路有关。     

Combination of tanshinone IIA and doxorubicin possesses synergism and attenuation effects on doxorubicin in the treatment of breast cancer

Doxorubicin (Dox) is a first‐line drug for breast cancer chemotherapy. However, with the prolongation of chemotherapy cycle, breast cancer cells are increasingly tempt to resist Dox, and meanwhile, high cumulative dose of Dox brings enhancing toxic side effects, and these effects may lead to chemotherapy failure. Hence, it is necessary to search an agent in combination medication with Dox, which can not only enhance the chemosensitivity of Dox but also reduce the toxic side effects. Tanshinone IIA (Tan IIA) is reported to have antitumor activity in addition to its cardiovascular protective effects. We employed human breast cancer MCF‐7 and MCF‐7/dox cells in order to assess whether Tan IIA might perform such function. Our in vitro studies showed that Tan IIA could enhance the sensitivity of breast cancer cells to Dox through inhibiting the PTEN/AKT pathway and downregulating the expression of efflux ABC transporters including P‐gp, BCRP, and MRP1. In addition, our in vivo studies showed Tan IIA enhanced the chemotherapeutic effect of Dox against breast cancer while reducing its toxic side effects including weight loss, myelosuppression, cardiotoxicity, and nephrotoxicity. Therefore, Tan IIA could be used as a novel agent combined with Dox in breast cancer therapy.     

Insight into the pharmacokinetic behavior of tanshinone IIA in the treatment of Crohn’s disease: comparative data for tanshinone IIA and its two glucuronidated metabolites in normal and recurrent colitis models after oral administration

1.?Previous reports implied that tanshinone IIA (TSA) may offer potential benefits for Crohn’s disease (CD). However, the detailed pharmacokinetic behavior of TSA in the treatment of colitis remain unclear. Herein, a recurrent trinitrobenzene sulfonic acid (TNBS)-colitis mouse model was used to investigate whether TSA possesses favorable pharmacokinetic and colonic distribution profiles to serve as a candidate drug.

2.?Although the systemic TSA exposures were low (AUC_0–t approximately 330?ng*h/ml) in both the normal and colitis models after oral administration TSA 20?mg/kg, high levels of TSA were found in the gastrointestinal tract (GI). Such a GI exposure of TSA in colitis mice is adequate to exert anti-inflammatory effects as observed in various in vitro studies.

3.?Interestingly, colonic TSA exposure in the colitis mouse model was much lower than that in the normal mice, which may be explained by a significant upregulation of colonic UDP-glucuronosyltransferase (Ugt)1a9 expression and a higher plasma concentration of TSA glucuronides in the model mice at 0.5, 1 and 2?h after TSA administration.

4.?Together, these results reveal high accumulation at the site of inflammation and minimal systemic concentration of TSA, which are favorable pharmacokinetic behaviors to meet the requirements for CD treatment.     

丹参酮对两种学习记忆功能障碍模型大鼠治疗作用的实验研究

目的研究丹参酮对拟人类阿尔茨海默病（AD）和血管性痴呆（VD）2种模型大鼠学习、记忆功能障碍的改善作用。方法①实验动物随机分成5组：痴呆（AD／VD）模型组、丹参酮治疗组、假造模对照组和正常对照组。VD模型组：应用改良的大脑中动脉线栓塞（MCAO）法建模;AD模型组：应用D-半乳糖腹腔注射和海马内注射β-淀粉样肽蛋白片段1-40（Aβ1-40）复合造模方法建模。丹参酮治疗组：VD大鼠造模完成后或AD大鼠造模24h后给予丹参酮[somg／（kg&#183;d）],溶于5ml玉米油中灌胃14d。假造模对照组：与VD大鼠同法,但不做插线、结扎;与AD大鼠同法注射等量溶媒。正常对照组：大鼠不做任何处理。②采用水迷宫行为学实验,以单位时间逃避潜伏期及其频率为指标,测定大鼠空间学习、记忆功能。结果①AD／VD大鼠逃避潜伏期明显延长,逃生错误频率高,与两个对照组比较差异均有显著性意义。②丹参酮治疗组大鼠较痴呆模型组逃避潜伏期明显缩短,逃生错误频率减低,差异有统计学意义。结论①2种痴呆大鼠模型具有较好仿真人类AD／VD的特点。②丹参酮对AD／VD具有一定的治疗作用。     

丹参酮ⅡA磺酸钠联合阿托伐他汀对冠心病患者外周血单个核细胞TLR4、MyD88、NF-κB表达的影响

目的：观察冠心病患者外周血单个核细胞 TLR4、MyD88、NF-κB 的表达,分析丹参酮ⅡA 磺酸钠联合阿托伐他汀对其表达的影响,以 TLR4炎症信号通路为靶点探讨丹参酮ⅡA 磺酸钠治疗冠心病的可能机制。方法选取诊治的冠心病患者160例,同期进行健康体检的正常人100例作为对照。采用 Real time-PCR 和 Western blot 测定外周血单个核细胞 TLR4、MyD88、NF-κB mRNA 和蛋白表达,采用酶联免疫吸附实验（ELISA）测定血清高敏 C-反应蛋白（hs-CRP）、肿瘤坏死因子-α（TNF-α）水平。160例冠心病患者随机分为观察 A 组和观察 B 组,每组80例。观察 A 组在常规治疗基础上给予阿托伐他汀治疗,观察 B 组在观察 A 组基础上加用丹参酮ⅡA 磺酸钠注射液,疗程均为30 d,比较2组 TLR4、MyD88、NF-κB mRNA 和蛋白表达、hs-CRP、TNF-α水平。结果与对照组比较,冠心病患者组 TLR4、MyD88、NF-κB mRNA 和蛋白表达、hs-CRP、TNF-α水平显著升高（ P ﹤0.05）。TLR4、MyD88、NF-κB 蛋白表达与 hs-CRP、TNF-α呈正相关（ r ＝0.761和0.802,0.699和0.774,0.835和0.749,P 均﹤0.05）。与治疗前比较,治疗后观察 A 组和观察 B 组 TLR4、MyD88、NF-κB mRNA 和蛋白表达、hs-CRP、TNF-α水平均显著降低（ P ﹤0.05）;但观察 B 组降低程度更明显（ P ﹤0.05）。结论丹参酮ⅡA 磺酸钠联合阿托伐他汀通过抑制外周血单个核细胞 TLR4炎症信号通路的激活发挥抗炎作用,可能是丹参酮ⅡA 磺酸钠发挥治疗作用的机制之一。