Impact of Linggui Zhugan decoction on microwave ablation outcomes and recurrence in liver cancer

BACKGROUND Liver cancer is one of the most common malignant tumors of the digestive system,and early detection and effective treatment are crucial for improving the prognosis.Microwave ablation(MWA)has shown promising results as a local therapeutic method for liver cancer;however,further improvement of its efficacy remains a key focus of current research.AIM To evaluate the clinical efficacy of Linggui Zhugan decoction combined with MWA for the treatment of primary liver cancer.METHODS Data were collected from 164 patients with primary liver cancer who underwent MWA at our hospital between March 2019 and April 2021.Among them,79 patients(control group)received routine treatments and 85 patients(research group)received Linggui Zhugan decoction in addition to routine treatment.The clinical efficacy,incidence of adverse reactions,and levels of serum alphafetoprotein(AFP),des-γ-carboxy prothrombin(DCP),AFP-L3,total bilirubin(TBil),alanine aminotransferase(ALT),CD4 cell count,CD8 cell count,and CD4/CD8 ratio were compared between the two groups,before and after treatment.The three-year recurrence rates between the two groups were compared,and independent prognostic factors for recurrence were identified.RESULTS The study results revealed that the objective response rate(ORR)in the research group was significantly higher than that in the control group(P=0.005).After treatment,the CD4 cell count and CD4/CD8 ratio significantly increased,whereas the CD8 cell count and TBil,ALT,AFP,DCP,and AFP-L3 Levels were significantly lower in the research group than in the control group(P<0.001).The Cox regression analysis revealed that the treatment regimen(P=0.003),presence of cirrhosis(P=0.019),tumor diameter(P=0.037),Child-Pugh score(P=0.003),pretreatment AFP level(P=0.006),and AFP-L3 Level(P=0.002)were independent prognostic factors for disease-free survival.CONCLUSION The combination of Linggui Zhugan decoction with MWA significantly improved the clinical efficacy and longterm prognosis of patients with primary liver cancer.

Linggui Zhugan Decoction Improves High Glucose-Induced Autophagy in Podocytes

Objective To explore the influence of Linggui Zhugan Decoction(LGZGD) on high glucose induced podocyte autophagy.Methods LGZGD containing serum was prepared by intragastric administation of 4.2 g/kg(low dose), 8.4 g/kg(medium dose), and 12.6 g/kg(high dose) LGZGD into SD rats respectively. MPC5 and AB8/13 podocyte cells were treated with 60 mmol/L glucose to establish diabetic nephropathy podocyte model in vitro. Both podocytes were divided into control group, high glucose group, low dose LGZGD group, medium dose LGZGD group, and high dose LGZGD group, respectively. For the three LGZGD groups, before LGZGD intervention, podocytes were treated with 60 mmol/L glucose for 3 days. After treated with LGZGD containing serum, cells were collected to analyze cell migration using Transwell assay, proliferation using CCK8, apoptosis and cell cycle using flow cytometry, autophagosome formation using transmission electron microscopy, and expression levels of Beclin-1, Atg5, LC3II/I, and P62 proteins using Western blot.Results Compared with the control group, the proliferation and migration of MPC5 and AB8/13 cells in the high glucose group slightly decreased, whereas these parameters restored after intervention with low and medium concentrations of LGZGD, with the medium dose LGZGD having the better effect(P < 0.05). Flow cytometry showed that the medium dose LGZGD group had a significantly lower apoptosis rate(P < 0.05) and higher survival rate(P > 0.05) compared to the high dose LGZGD group. High glucose arrested podocytes in G1 phase, whereas LGZGD shifted podocytes from being predominant in G1 phase to G2 phase. High dose LGZGD significanly reduced high glucose-increased autophagosome formation in both podocytes(P < 0.05). Western blot analysis showed that Beclin-1, Atg5, LC3II/I, and P62 expressions were increased in MPC5 cells treated with high glucose and reversed after adminstration of low and medium doses of LGZGD(P < 0.05).Conclusion LGZGD reduced apoptosis and enhanced autophagy in high glucose treated podocytes via regulating Beclin-1/LC3II/I/Atg5 expression.

Pharmacodynamic study and mechanism of action of Linggui Zhugan Decoction in the intervention of Nonalcoholic fatty liver disease

Objective: To explore the therapeutic effect of Linggui Zhugan Decoction on nonalcoholic fatty liver disease through in vivo animal experiments, and to explore the mechanism of action of Linggui Zhugan Decoction in the intervention of nonalcoholic fatty liver disease through network pharmacology and molecular docking technology. Methods: A rat model of non- alcoholic fatty liver disease was established after 20 weeks of high-fat feeding, and the rats were divided into six groups, normal group, model group, simvastatin group (4 mg/kg), low- dose group of Linggui Zhugan Decoction (2.1 g/kg), medium-dose group of Linggui Zhugan Decoction (4.2 g/kg), and high-dose group of Linggui Zhugan Decoction (8.4 g/kg), and the liver morphological changes of HE staining and oil red O staining after the intervention of Linggui Zhugan Decoction were observed, and the microplate reader detected aspartate aminotransferase, alanine aminotransferase, Four levels of blood lipids. Network pharmacology combined with molecular docking was used to screen the potential mechanism and target of Linggui Zhugan Decoction for the intervention of non-alcoholic fatty liver disease. Results: Linggui Zhugan Decoction significantly improved the levels of alanine aminotransferase and alanine aminotransferase in model rats (P<0.05). HE staining and oil red O staining showed that Linggui Zhugan Decoction significantly reduced liver lipid deposition in rats in the model group. Linggui Zhugan Decoction could significantly reduce the quadruplical levels of blood lipids in model rats (P<0.05). Network pharmacology screened out 10 key targets and 5 key signaling pathways;The molecular docking results showed that the first 20 active ingredients all had good binding ability to the top 10 targets. Conclusion: The results show that Linggui Zhugan Decoction has the effect of regulating blood lipids and improving liver tissue lesions in non-alcoholic fatty liver disease, and preliminarily verifies the regulatory effect of Linggui Zhugan Decoction on lipid metabolism-related genes.

Linggui Zhugan Decoction(苓桂术甘汤)Inhibits Ventricular Remodeling after Acute Myocardial Infarction in Mice by Suppressing TGF-β1 Smad Signaling Pathway

Objective:To investigate the inhibitory effect of Linggui Zhugan Decoction(LZD,苓桂术甘汤)on the ventricular remodeling(VR)after acute myocardial infarction(AMI)and related mRNA and proteins expression in transforming growth factor-beta 1(TGF-β1)/Smad signaling pathway,and explain its putative mechanism.Methods:A VR model was generated by ligation of coronary artery in mice.Two weeks after surgery,60 mice were randomly divided into the model group,the sham-operation group(distilled water),the positive control group(2.4 mg/kg simvastatin),and the low-,medium-and high-dose LZD groups(2.1,4.2,8.4 g crude drug/kg,respectively)by a random number table,10 mice in each group.Mice in each group was treated for 4 weeks.Changes of hemodynamics indices and cardiac weight index were detected by the PowerLab data acquisition and analysis recording instrument.Morphology changes of myocardial tissue were observed by hematoxylin-eosin and Masson staining.The expressions of TGF-β1,Smad2,Smad3,p-Smad2 and p-Smad3 in myocardial tissue were detected by Western blotting.The mRNA expressions of TGF-β1,Smad2 and Smad3 were detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR).The expressions of matrix metalloprotein 2(MMP2),MMP9,collagenⅠand collagen Ⅲ were observed by immunohistochemical methods.Results:VR mice showed significant dysfunction in hemodynamic indices and cardiac structure and function.Compared with the shamoperation group,myocardial tissue damage,interstitial fibrosis occurred in the model mice,left ventricular systolic pressure(LVSP),left ventricular pressure maximum contraction rate(+dp/dtmax)and left ventricular pressure maximum relaxation rate(-dp/dtmax)decreased significantly(all P<0.01),while left ventricular end-diastolic pressure(LVEDP),cardiac weight index and left ventricular weight index elevated significantly,meanwhile TGF-β1,p-Smad2,p-Smad3,Smad2,Smad3,MMP2,MMP9,collagen Ⅰ,collagen Ⅲ protein expressions in myocardial tissue and TGF-β1 Smad2 and Smad3 mRNA expressions increased significantly(all P<0.01).Compared with the model group,LZD could significantly improve the pathological changes of myocardial tissue,increase LVSP,+dp/dtmax and-dp/dtmaxf lower LVEDP,reduce the whole heart weight index and left ventricular weight index and inhibit the over-expressions of TGF-β1f p-Smad2,p-Smad3,Smad2,Smad3,MMP2,MMP9,collagenⅠand collagenⅢproteins in myocardial tissue and mRNA expressions of TGF-β1 Smad2 and Smad3(P<0.05 or P<0.01).Conclusion:LZD can significantly suppress VR induced by AMI,and its underlying mechanism may be associated with its inhibitory effect on the TGF-β1/Smad signaling pathway.

Effects of the Modified Linggui Zhugan Decoction(加味苓桂术甘汤) Combined with Short-term Very Low Calorie Diets on Glycemic Control in Newly Diagnosed Type 2 Diabetics

Objective:To evaluate the effects of the modified Linggui Zhugan Decoction(加味苓桂术甘汤) combined with short-term very low calorie diets(VLCDs) on glycemic control in newly diagnosed type 2 diabetes mellitus(T2DM) patients.Methods:A total of 20 subjects with newly diagnosed T2DM were treated with the modified Linggui Zhugan Decoction(one-month administration) combined with short-term VLCDs(5 days),and 3-months follow-up.A standard 75-g oral-glucose-tolerance test(OGTT) indexes fasting plasma glucose(FPG),post-prandial 0.5 h and 2 h plasma glucose(P0.5hPG,P2hPG),glycated hemoglobin A1C(GHbA1C),body weight,body mass index(BMI),insulin function,insulin resistance index,incidence of hypoglycemia,and the liver and renal functions were evaluated before and after treatment.Correlations of BMI with insulin function and insulin resistance were also assessed.Results:After the treatment,the patients' plasma glucose decreased steadily,FPG decreased from 5.8±0.9 mmol/L at pre-treatment to 5.0±0.6 mmol/L at 3-months follow-up(P<0.05),and P2hPG decreased from 11.7±3.8 mmol/L at pre-treatment to 6.9±0.9 mmol/L at 3-months follow-up(P<0.01).The level of GHbA1C declined from(6.47±1.24)% at pre-treatment to(6.14±0.99)% at 3-months follow-up(P<0.01).Body weight and BMI also declined significantly.Insulin resistance index was improved obviously and no event of hypoglycemia occurred.Part of the patients companied with fatty liver had a transient increase in hepatic transaminase during the treatment,but it turned to normal after the treatment.Conclusions:The modified Linggui Zhugan Decoction combined with short-term VLCDs can be safely implemented for steady glycemic control in newly diagnosed T2DM patients.

Protective effects of Modified Linggui Zhugan Decoction combined with short-term very low calorie diets on cardiovascular risk factors in obese patients with impaired glucose tolerance

OBJECTIVE:To observe the protective effects of modified Linggui Zhugan Decoction combined with short-term very low calorie diets(VLCDs) on cardiovascular risk factors in obese patients with impaired glucose tolerance(IGT).METHODS:Eighty-five obese patients with IGT of spleen hypofunction and dampness accumulation type were randomly divided into treated group(n=45)and control group(n=40).The treated group was given Linggui Zhugan Decoction combined short-term VLCDs.The control group was given basic weight-reduction treatment.The total course was 6 months.Changes of fasting plasma glucose(FPG),2-hour postprandial blood glucose(2hPG),glycosylated hemoglobin(HbA1c),fasting insulin(FINS),lipid metabolism,blood pressure,body mass index(BMI) and waist circumference(WC) were observed,and the outcomes were reviewed after the treatment.RESULTS:Compared with the control group,the levels of FPG,2hPG,HbA1c,FINS,HOMA-IR decreased significantly(P<0.05),and the indexes of total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL),systolic blood pressure(SBP),diastolic blood pressure(DBP),BMI and WC were improved significantly(P<0.05) in the treated group.The transformation rate of normal glucose tolerance(NGT) was higher in treatment group than control group(P<0.01).No severe adverse reaction was found in the therapeutic course.CONCLUSION:The modified Linggui Zhugan Decoction combined with short-term VLCDs has good protective effects on cardiovascular risk factors as a treatment intervention for IGT with obesity,as it could improve glycometabolism,significantly decrease the levels of blood pressure,blood lipids,BMI,WC and effectively cut-off and reverse the development of diabetes mellitus.

Identification of Cald1 as a novel regulator of Linggui Zhugan decoction(苓桂术甘汤) for improving insulin resistance in vivo and in vitro

OBJECTIVE:To identify Cald1 as a novel regulator of Linggui Zhugan decoction(苓桂术甘汤)for improving insulin resistance in vivo and in vitro.METHODS:Sprague-Dawley rats were randomly assigned to 3 groups that were received a normal rat chow diet,high-fat diet(HFD),and an HFD plus LGZGD,respectively.The homeostatic model assessment(HOMA)-insulin resistance(IR)index was used to determine IR.Gene microarray methodology was used to identify differentially expressed genes(DEGs)in the three groups of rats.The DEGs associated with IR were confirmed by quantitative real-time polymerase chain reaction.Additionally,Mouse 3 T3-L1 pre-adipocytes were differentiated into mature 3 T3-L1 adipocytes,which were then treated with tumor necrosis factor(TNF)-αto induce cellular IR.Lipid accumulations were identified by Oil Red O staining.Glucose uptake was assessed using the3 H-2-DG test.RESULTS:In this study,we found Cald1 was further screened to validate its biological function in3 T3-L1 adipocytes induced to develop IR.In vitro experiments showed that insulin-stimulated 3 H-2-DG uptake by IR 3 T3-L1 adipocytes was increased after LGZGD intervention,which was associated with a down-regulation of Cald1 expression.CONCLUSION:LGZGD ameliorates HFD-induced IR in rats and TNF-αinduced IR in adipocytes by down-regulating Cald1 expression.

加味苓桂术甘汤对阳虚水停证慢性心力衰竭患者TGF-β1、TNF-α、NT-proBNP及心室重构的影响

目的探究加味苓桂术甘汤对阳虚水停证慢性心力衰竭患者TGF-β1、TNF-α、NT-proBNP及心室重构的影响。方法将2017年12月至2020年11月六安市中医院收治的98例阳虚水停证慢性心力衰竭患者纳入本研究,随机分为两组,各49例。对照组行西医常规治疗,观察组在此基础上行加味苓桂术甘汤口服治疗,治疗6个月后检测心功能,血清肿瘤坏死因子(tumor necrosis factor,TNF)-α、转化生长因子(transforming growth factor,TGF)-β1、同型半胱氨酸(homocysteine,Hcy)、氨基末端脑钠素原(amminoterminal brain sodium minogen,NT-proBNP)水平并比较临床疗效。结果两组患者治疗后各项指标均较治疗前明显改善(P<0.05)。其中观察组患者治疗后血清TNF-α、TGF-β1、Hcy、NT-proBNP、左室舒张末期内径、左室收缩末期内径和中医证候总积分低于对照组,左室射血分数、6 min步行距离和临床疗效高于对照组(P<0.05)。结论加味苓桂术甘汤治疗阳虚水停证慢性心力衰竭可减轻患者炎症反应,预防心室重构,提高临床疗效。

从“微饮”立论射血分数保留的心力衰竭早期防治思想

射血分数保留的心力衰竭(heart failure with preserved ejection fraction, HFpEF)是目前心血管领域的治疗难点,防治前线前移对于减缓或逆转HFpEF发病进程至关重要。聚焦HFpEF早期的中医防治,是发挥中医治未病优势的关键。通过梳理仲景提出的“微饮”内涵和证治,结合HFpEF早期的发病机制,提出HFpEF早期以微饮内停上焦为主要病机,临证应以苓桂术甘汤为主祛饮通阳,以期阻断饮证进一步发展,截断HFpEF发展进程。从“微饮”立论HFpEF早期防治,可以防微杜渐,具有重要的临床意义。

经典名方苓桂术甘汤古籍考证与分析

苓桂术甘汤是2018年国家中医药管理局公布的《古代经典名方目录(第一批)》中收载的经典名方之一。通过收集整理古籍信息,从处方历史沿革、功能主治、药品炮制、药物剂量折算等方面,对苓桂术甘汤进行考证。经考证,苓桂术甘汤在《伤寒论》和《金匮要略》中均有记载,且药物配比及炮制方法略有差异,经典名方开发宜遵照《金匮要略》中药物剂量配比,桂枝使用《中华人民共和国药典》2020版中所规定的“桂枝”,并按其项下内容进行炮制,甘草宜用“炒甘草”。功能主治在水湿痰饮之病因的基础上有所延伸,包括阳虚水盛、痰饮咳嗽呕水等。药物剂量折算可以依据汉代一两等于13.8 g进行折算。

苓桂术甘汤干预非酒精性脂肪肝病的药效学研究及作用机制探讨

目的:通过体内动物实验探究苓桂术甘汤对非酒精性脂肪肝病的治疗效果,网络药理学及分子对接技术探究苓桂术甘汤干预非酒精性脂肪肝病的作用机制。方法:高脂喂养20周建立非酒精性脂肪肝病大鼠模型,将大鼠分为6组,正常组、模型组、辛伐他汀组(4 mg/kg)、苓桂术甘汤低剂量组(2.1 g/kg)、苓桂术甘汤中剂量组(4.2 g/kg)、苓桂术甘汤高剂量组(8.4 g/kg),观察苓桂术甘汤干预后的HE染色及油红O染色的肝脏形态学变化,酶标仪检测血清中谷草转氨酶、谷丙转氨酶、血脂四项水平。利用网络药理学结合分子对接筛选苓桂术甘汤干预非酒精性脂肪肝病潜在作用机制及靶点。结果:苓桂术甘汤可显著改善模型大鼠的谷草转氨酶、谷丙转氨酶水平(P<0.05);HE染色和油红O染色显示苓桂术甘汤显著降低模型组大鼠的肝脏脂质沉积;苓桂术甘汤可显著降低模型大鼠血脂四项水平(P<0.05);网络药理学筛选出关键靶点10个,关键信号通路5条;分子对接结果表明前20活性成分均对前十靶点有良好结合能力。结论:表明苓桂术甘汤对非酒精性脂肪肝病有调节血脂及改善肝组织病变作用,并初步验证了苓桂术甘汤对脂质代谢相关基因的调控作用。

基于网络药理学和分子对接技术探讨苓桂术甘汤改善非酒精性脂肪性肝病的潜在作用机制

目的:通过网络药理学和分子对接技术预测和探讨苓桂术甘汤改善非酒精性脂肪肝病的活性成分及其潜在的作用机制。方法:采用TCMSP、ETCM数据库筛选出苓桂术甘汤的活性成分和相关的潜在靶点。利用GeneCards数据库检索非酒精性脂肪肝病相关靶点。基于苓桂术甘汤潜在靶点与非酒精性脂肪肝病相关靶点的匹配结果,在STRING平台进行蛋白质相互作用分析,筛选核心靶点。利用CytoScape 3.9.1软件拓扑分析筛选出关键活性成分,构建苓桂术甘汤活性成分-非酒精性脂肪肝病-靶点-通路网络。通过Reactome与Metascape数据库进行基因本体论(GO)与京都基因与基因组百科全书(KEGG)富集分析。利用Autodock Tools 1.5.7将前5位核心靶点和对应的活性成分进行分子对接。结果:苓桂术甘汤改善非酒精性脂肪肝病的重点活性成分为槲皮素、柚皮素、山柰酚等,关键靶点有细胞肿瘤抗原p53(Cell Tumor Antigen p53,TP53)、肿瘤坏死因子(Tumor Necrosis Factor,TNF)、白细胞介素-1β(Interleukin-1β,IL-1β)、白细胞介素-6(Interleukin-6,IL-6)、丝氨酸/苏氨酸蛋白激酶1(Akt Serine/Threonine Kinase 1,AKT1),主要涉及细胞对脂质的反应、对异种刺激的反应、无机物质反应等生物过程,主要富集在癌症通路、糖尿病并发症中的晚期糖基化终产物及其受体信号通路、白细胞介素-17信号通路等。TP53、TNF、IL-6、IL-1β与槲皮素对接良好,AKT1与柚皮素对接良好。结论:苓桂术甘汤可以多方向、多维度改善非酒精性脂肪肝病,本研究为进一步揭示苓桂术甘汤改善非酒精性脂肪肝病机制提供了理论基础和参考依据。

苓桂术甘汤干预FXR途径影响游离脂肪酸诱导的HepG2细胞脂质代谢

目的 观察苓桂术甘汤对FFA诱导的HepG2细胞脂质代谢的影响,探讨其作用机制是否与其干预FXR途径有关。方法 选用HepG2细胞,采用1mmol/L FFA诱导,实验分为正常组、模型组、苓桂术甘汤组、阿托伐他汀组。采用CCK-8法测定细胞活力,油红O染色法观察脂滴分布,生物试剂盒测定TG、TC含量,蛋白免疫印迹法检测FXR、SHP、CYP7A1、LXR、SREBP-1蛋白的表达量,免疫荧光法检测FXR的荧光表达强度。结果 与正常组相比,模型组脂滴分布增加,TG、TC含量升高(P<0.01),FXR、SHP蛋白表达水平降低,CYP7A1、LXR和SREBP-1蛋白表达水平升高,FXR荧光表达水平降低;与模型组比较,苓桂术甘汤组和阿托伐他汀组脂滴分布下降,TG、TC含量降低(P<0.01),FXR、SHP蛋白表达水平升高,CYP7A1、LXR和SREBP-1蛋白表达水平降低,FXR荧光表达水平升高。结论 苓桂术甘汤可显著抑制游离脂肪酸诱导的HepG2细胞脂肪沉积,其作用机制可能与干预FXR介导的脂质代谢通路有关。

苓桂术甘口服液澄清工艺及抗脂肪肝作用初步研究

目的 优选苓桂术甘口服液的澄清工艺,初步探讨其抗脂肪肝作用。方法 比较水提醇沉法、澄清剂法、高速离心法、膜过滤法、热处理法5种澄清工艺对苓桂术甘口服液中甘草酸含量及澄明度的影响,以综合评分的方法确定最佳澄清工艺。测定小鼠体质量变化,采用苏木素-伊红染色和油红O染色探讨肝脏脂滴情况。结果 经过比较苓桂术甘口服液的不同澄清工艺,得出ZTC1+1澄清剂法的综合评分最高,因此选择ZTC1+1澄清剂法为苓桂术甘口服液的最佳澄清工艺。模型组小鼠体质量大于空白组,口服液干预后体质量显著下降,其中,高剂量组苓桂术甘口服液降低最为显著。苏木素-伊红染色结果发现,与模型组相比,治疗组脂滴空泡明显减少,肝细胞肿胀程度降低;油红O染色结果发现,与模型组相比,治疗组小鼠肝组织脂质沉积明显减少。结论 苓桂术甘口服液最佳澄清工艺为ZTC1+1澄清剂法,澄清剂用量为4%︰2%,药液浓度为1︰4,絮凝温度为70 ℃。苓桂术甘口服液能够降低体质量、减少小鼠肝脏脂滴沉积,初步证明其具有抗脂肪肝作用。

苓桂术甘汤调节PKA/CREB通路改善阿尔茨海默病小鼠海马神经元突触损伤的作用研究

目的观察苓桂术甘汤对阿尔茨海默病(Alzheimer's disease,AD)小鼠海马神经元突触损伤的改善作用,并探讨其作用机制。方法采用双侧海马注射Aβ1-42法建立AD小鼠模型,造模成功后随机分为AD组,苓桂术甘汤低、中、高剂量组(4.29、21.45、42.90 g/kg)及石杉碱甲组(0.04 mg/kg),每组各10只。灌胃给药4周后,Morris水迷宫检测各组大鼠学习记忆能力;透射电镜观察海马区突触超微结构变化并测量突触后致密带厚度;HE染色观察海马组织病理变化;Western blot检测海马组织蛋白激酶A(protein kinase A,PKA)、cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)、磷酸化-CREB(phosphorylation-CREB,p-CREB)蛋白表达。结果与AD组比较,高、中、低剂量组大鼠逃避潜伏期明显缩短,穿越原隐藏平台次数明显增加,目标象限停留时间明显延长,海马神经元突触后致密带厚度明显增大,海马组织PKA蛋白表达量、p-CREB/CREB明显升高(P<0.05)。结论苓桂术甘汤可能通过激活PKA/CREB信号通路减轻海马神经元突触损伤,进而改善AD小鼠的学习记忆能力。

基于UPLC指纹图谱结合化学计量学的经典名方苓桂术甘汤质量评价研究

目的建立指纹图谱与化学计量学相结合的经典名方苓桂术甘汤的质量评价方法。方法使用SHIMADZU Shim-Pack GIST C18色谱柱(100 mm×2.1 mm,2.0μm),流动相为乙腈-0.1%磷酸水溶液,梯度洗脱,柱温30℃,流速0.2 mL/min,检测波长为266 nm(前30 min)、235 nm(后36 min)。使用《中药色谱指纹图谱相似度评价系统》(2012.130723版)建立苓桂术甘汤超高效液相色谱指纹图谱,确定共有峰并进行相似度评价;基于指纹图谱共有峰峰面积测定结果,采用聚类分析、主成分分析和正交偏最小二乘-判别分析(OPLS-DA)对不同批次苓桂术甘汤进行质量评价。结果共确认苓桂术甘汤指纹图谱24个共有峰,使用对照品指认14个共有峰,10批苓桂术甘汤样品相似度均大于0.950。10批苓桂术甘汤可聚为两类,主成分1~4是影响其质量评价的主要因子,OPLS-DA共确定6个差异标志物。结论本研究建立的指纹图谱方法简便可靠,重复性良好,指纹图谱结合化学计量学分析可明确不同产地药材制成的苓桂术甘汤之间的差异,为其内在质量评价提供参考。

基于氧化应激及其相关凋亡蛋白研究苓桂术甘汤预处理对大鼠心肌缺血再灌注损伤的保护作用及机制

目的 基于氧化应激及其相关凋亡蛋白探讨苓桂术甘汤对大鼠心肌缺血再灌注损伤的保护作用及机制。方法 45只SPF级SD雄性大鼠,适应性喂养1周,随机分成假手术组(Sham组)、心肌缺血再灌注组(IR组)、苓桂术甘汤预处理组[IR+LGZG(0.68 g/kg)组],每组15只。依据组别灌胃给予相应药物预处理,1次/d,连续7 d。第8天通过结扎大鼠心脏冠状动脉左前降支(ligating the anterior descending, LAD),心肌缺血30 min后,进行缺血再灌注24 h, Sham组只做穿线处理不结扎,建立MIRI大鼠模型。造模成功后分别取各组大鼠的腹主动脉血清和心脏组织进行相关指标的检测。通过心电图观察心功能变化,通过TTC染色观察心肌梗死面积,通过HE染色观察心肌组织病理改变。采用生化和ELISA法检测心肌酶损标志物Tn-T、CK-MB、LDH含量。采用ELISA法检测血清中氧化应激指标SOD、MDA、GSH-Px含量。采用Western blot法检测心肌组织中的凋亡蛋白Bax、Caspase-3、Bcl-2含量。结果 与Sham组相比,IR组大鼠的心电图ST段向上拱起明显,心肌组织病理形态学发生改变,心肌梗死面积显著增加(P<0.01),血清中Tn-T、CK-MB、LDH浓度显著升高(P<0.01),SOD表达显著降低(P<0.01),MDA表达显著升高(P<0.01),心肌细胞中Caspase-3、Bax蛋白表达上调(P<0.01),Bcl-2表达下调(P<0.01);与IR组相比,IR+LGZG组可以减少MIRI所致心脏损伤(P<0.05),升高SOD的表达(P>0.05),降低MDA的表达(P<0.01),减少MIRI引起的心肌细胞凋亡(P<0.05)。结论 苓桂术甘汤对大鼠心肌缺血再灌注损伤治疗作用佳,其作用机制部分是通过抑制氧化应激和随后的心肌细胞凋亡来实现的。

苓桂术甘汤调控miR-140-5p/TLR4/NF-κB信号通路对溃疡性结肠炎大鼠肠黏膜屏障及免疫平衡的影响

目的:探讨苓桂术甘汤调控miR-140-5p/Toll样受体4(toll like receptor 4,TLR4)/核转录因子-κB(nuclear factor-kappa B,NF-κB)信号通路对溃疡性结肠炎(ulcerative colitis,UC)大鼠肠黏膜屏障及免疫平衡的影响。方法:取SD大鼠用2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzenesulfonic acid solution,TNBS)诱导建立UC模型,随机分为5组:模型组、苓桂术甘汤低剂量组、苓桂术甘汤高剂量组、NC-miR-140-5p(miR-140-5p阴性对照)组、苓桂术甘汤高剂量组+miR-140-5p antagomir(miR-140-5p抑制剂)组,每组10只,另取10只大鼠作对照组,分组干预后检测大鼠结肠黏膜损伤指数(colonic mucosal injury index,CMDI)评分与结肠长度;透射电镜观察各组大鼠结肠黏膜屏障超微结构;流式细胞实验检测各组大鼠外周血Treg、Th17细胞比例及Treg/Th17比值;ELISA检测大鼠血清免疫炎症相关因子白细胞介素(interleukin,IL)-6、IL-17、IL-10、转化生长因子β1(transforming growth factor-β1,TGF-β1)水平;采用实时荧光PCR及免疫印迹法检测大鼠结肠黏膜组织miR-140-5p/TLR4/NF-κB信号相关因子表达。结果:与对照组比较,模型组大鼠结肠黏膜屏障超微结构损伤严重,CMDI、Th17细胞比例、血清IL-6与IL-17水平、结肠黏膜组织TLR4 mRNA、蛋白表达与p-NF-κB p65/NF-κB p65显著升高(P<0.05),结肠长度、Treg细胞比例、Treg/Th17比值、血清IL-10与TGF-β1水平、结肠黏膜组织claudin-1、claudin-4蛋白表达与miR-140-5p表达显著降低(P<0.05)。与模型组比较,苓桂术甘汤低剂量组、苓桂术甘汤高剂量组大鼠结肠黏膜屏障超微结构损伤均减轻,CMDI、Th17细胞比例、血清IL-6与IL-17水平、结肠黏膜组织TLR4 mRNA、蛋白表达与p-NF-κB p65/NF-κB p65均降低(P<0.05),结肠长度、Treg细胞比例、Treg/Th17比值、血清IL-10与TGF-β1水平、结肠黏膜组织claudin-1、claudin-4蛋白表达与miR-140-5p表达均升高(P<0.05);NC-miR-140-5p组大鼠各指标差异无统计学意义(P>0.05);高剂量苓桂术甘汤作用更强。下调miR-140-5p可减弱高剂量苓桂术甘汤对UC模型大鼠各指标的作用。结论:苓桂术甘汤可通过上调miR-140-5p而抑制TLR4/NF-κB信号激活,进而促使Treg/Th17免疫平衡向Treg偏移,抑制免疫炎症,减轻UC大鼠肠黏膜屏障损伤。

通元针法联合加味苓桂术甘汤治疗脾虚痰湿型多囊卵巢综合征的临床观察

【目的】观察通元针法联合加味苓桂术甘汤治疗脾虚痰湿型多囊卵巢综合征(PCOS)的临床疗效。【方法】将86例脾虚痰湿型PCOS患者随机分为观察组和对照组,每组各43例。对照组给予盐酸二甲双胍联合炔雌醇环丙孕酮片治疗,观察组在对照组治疗的基础上,给予通元针法联合加味苓桂术甘汤治疗,2组患者均持续治疗3个月。治疗3个月后,评价2组临床疗效,观察2组患者治疗前后性激素指标雌二醇(E2)、睾酮(T)、促黄体生成激素(LH)、促卵泡生成激素(FSH)、催乳激素(PRL)表达水平的变化情况,比较2组患者治疗前后糖代谢水平包括胰岛素抵抗指数(HOMA-IR)、空腹胰岛素(FINS)、空腹血糖(FPG)表达水平的变化情况。【结果】(1)观察组总有效率为90.70%(39/43),对照组为74.42%(32/43)。观察组疗效优于对照组,差异有统计学意义(P<0.05)。(2)治疗后,2组患者的HOMA-IR、FINS、FPG水平均明显改善(P<0.05),且观察组在改善HOMA-IR、FINS、FPG水平方面明显优于对照组,差异有统计学意义(P<0.05)。(3)治疗后,2组患者E2、T、LH、FSH、PRL水平均明显改善(P<0.05),且观察组在改善患者E2、T、LH、FSH、PRL水平方面明显优于对照组,差异均有统计学意义(P<0.05)。【结论】通元针法联合加味苓桂术甘汤治疗脾虚痰湿型PCOS,可有效改善患者性激素水平,调节患者机体糖代谢,临床疗效显著。

心宝丸与苓桂术甘汤合生脉饮加减方治疗缓慢型心律失常的效果观察

目的 分析缓慢型心律失常患者实施心宝丸+苓桂术甘汤合生脉饮加减方治疗的效果。方法 120例缓慢型心律失常患者,以入院单双号分组,即对照组、研究组,每组60例。对照组给予心宝丸治疗,研究组在对照组治疗基础上辅以苓桂术甘汤合生脉饮加减方治疗。比较两组患者心率状态、转归情况、临床时间指标、临床疗效、不良反应发生情况。结果 治疗后,两组患者室性期前收缩频率较治疗前均降低、且研究组室性期前收缩频率(1904.51±80.02)次/d低于对照组的(2369.21±99.18)次/d,两组患者心脏搏动频率较治疗前均上升、且研究组心脏搏动频率(61.09±2.68)次/min高于对照组的(55.73±2.04)次/min(P<0.05)。治疗后,两组左心室射血分数(LVEF)均较治疗前升高、且研究组LVEF(61.26±2.95)%高于对照组的(53.80±2.11)%,两组患者症状积分均较治疗前降低、且研究组症状积分(0.97±0.26)分低于对照组的(1.48±0.32)分(P<0.05)。研究组起效时间(3.49±1.09)d、心电图恢复时间(14.28±2.53)d、出院时间(15.91±1.66)d均短于对照的(5.53±2.31)、(18.09±2.77)、(20.05±2.12)d(P<0.05)。研究组临床总有效率达到96.67%,对照组仅为81.67%,研究组高于对照组(P<0.05)。研究组、对照组不良反应发生率未表现出差异(P>0.05)。结论 心宝丸+苓桂术甘汤合生脉饮加减方治疗缓慢型心律失常有显著疗效,能迅速恢复心电图状态,提升心功能,恢复心脏搏动频率,且安全性高,对缓慢型心律失常较适用。