Acute Toxicity Test of Chinese Herbal Compound Tongmai Tangyanming Capsule

[Objective] This study was conducted to determine the safe dose of Tongmai Tangyanming capsule in clinic by evaluating its acute toxicity, so as to ensure clinical medication safety. [Method] The test was designed according to ＂Technique Requirements of Research of New Chinese Medicine＂ and ＂Guide for Research of New Chinese Medicine＂, mice were intragastrically administrated with Tongmai Tangyanming capsule at a maximum concentration and a maximum volume, and the acute toxic response of mice was observed, so as to determine medi- na lethal dose （LDso） and maximum administration dosage of Tongmai Tangyanming capsule. [Result] The LD^o could not be detected in the test, and the maximum administration dosage of mouse was calculated to be 112.104 g of dried herbs/（kg.d）, equivalent to 260 times of clinical dose of adult. [Conclusion] Chinese herbal compound Tongmai Tangyanming capsule has low acute toxicity, and its clinical dose is safe and reliable.

Effect of Tongmai Jiangzhi Oral Liquid (通脉降脂口服液) on Serum P-selectin, von Willebrand Factors and D-Dimer in Patients with Atherosclerosis

Objective: To explore the role of cytokines on the pathogenesis of atherosclerosis, and the effect of Tongmai Jiangzhi oral liquid (通脉降脂口服液,TMJZ) on cytokines through observing serum P-se-lectins (Ps), von Willebrand (vWF), and D-dimer (D-D) in atherosclerosis (AS) patients. Methods: Sixty-three AS patients were randomly divided into the treated group (n = 33, treated with TMJZ, 10 ml each time, three times a day) and the control group (n = 30, treated with Lovastatin, 10 mg, once daily). The levels of serum lipids (enzymatic methods), Ps, vWF, and D-D were measured before and after 8 weeks of treatment. Results: Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), apo-protein B-100 (apoB-100) were significantly decreased (P<0.05 or P< 0.01) and high-density lipoprotein cholesterol (HDL-C) was significantly raised (P<0.05) after TMJZ treatment. Serum Ps, vWF, and D-D also declined (P<0.05) after treatment. There was no significant difference between the treated and the control groups in these parameters except serum HDL-C level. Conclusion: TMJZ has good therapeutic effect in regulating serum lipids, improving endothelial cell function, inhibiting activation of platelets, and preventing the disturbance of blood coagulation/fibrinolysis function in patients with AS.

Effect of acupuncture combined with Xijing Tongmai decoction on the sustained expression of transient outward potassium channel related protein in rats with myocardial infarction

Objective:To observe the sustained expression of transient outward potassium channel related proteins at the end of the treatment and 30 days after the end of the treatment in rats,and to explore the sustained curative effect and mechanism of acupuncture combined with Xijingtongmai decoction in rats with myocardial infarction.Methods:Twenty of 130 male SD rats were random extracted as the control group,and the rest were used to establish myocardial infarction by fed with high-fat diet and then injected with isoproterenol.According to ECG,80 rats were successfully established.Then they were randomly divided into model group,acupuncture combined with Chinese medicine group,acupuncture group and Western medicine group.The content of bFGF protein was measured by ELISA.The protein contents of Kv1.4,Kv4.2,Kv4.3 and KChIP2 were measured by Western blot.Results:At the end of treatment,compared with the model group,the protein contents of Kv1.4,Kv4.2,Kv4.3,KChIP2 and bFGF in each treatment group increased,and the increase was most significant in the acupuncture combined with Chinese medicine group(P<0.05).At the end of treatment,compared with the model group,the protein contents of Kv1.4,Kv4.2,Kv4.3,KChIP2 and bFGF in each treatment group increased,and the increase was most significant in the acupuncture combined with Chinese medicine group(P<0.05).Compared with the treatment group at the end of treatment,the expression of Kv1.4,Kv4.2,Kv4.3,KChIP2 and bFGF protein in each treatment group 30 days after the end of treatment decreased slightly(P<0.05),but still higher than that of the model group at this time(P<0.05).The combination of acupuncture and Chinese medicine group decreased the least of them(P<0.05).Conclusion:The results showed that acupuncture combined with xijingtongmai decoction had a sustained good effect.Its sustained action mechanism may be achieved by continuously increasing the protein content of Kv1.4,Kv4.2,Kv4.3,KChIP2 and bFGF through transient outward potassium channel.

Effect of Tongmai Decoction on expression of NF-κB in ox-LDL injured endothelial cells

Objective Study on the effect of NF-κB in ox-LDL injured HUVEC-304 and the intervention with the serum of Tongmai Decoction. Methods Tongmai Decoction herbage-contained serum was made by the serum pharmacology methods, then the HUVEC-304 cells were divided into 5 groups and incubated for 24 h: 1) normal group;2) fetal calf serum group; 3) ox-LDL group; 4) simvastatin group; 5) Tongmai Decoction group. RT-PCR was used to determine the mRNA of NF-κB in the cells, Western Blot Assay was used to determine the protein content of NF-κB in the cells. Results 1) Compared with the cells in normal group and serum control group , the endothelial cells in ox-LDL group are seriously injured, while those in Tongmai Decoction group grow better; 2) Compared with the cells in normal group and fetal calf serum group, the expressions of NF-κB gene and protein are up-regulated in ox-LDL group(P＜0.05, P＜0.01); 3) Compared with the cells in ox-LDL group, the expression of NF-κB gene and protein in Tongmai Decoction group are significantly down-regulated, there is no difference between Tongmai Decoction group and simvastatin group(P＞0.05). Conclusion Tongmai Decoction can protect the injured endothelial cells, up-regulate the expressions of NF-κB gene and protein content.

Effects of Different Material Ratios on the Dissolution of Tongmai Pills

[Objectives]To study the effects of different material ratios on the dissolution of Tongmai pills.[Methods]Based on mixture uniform experiment design,the fiber,starch and grease-resin materials in Tongmai pills were proportioned.The contents of strychnine,brucine,paeoniflorin,calycosin,ferulic acid,hesperidin and salvianolic acid B in the solution were determined by HPLC,and the content of total flavonoids was determined by ultraviolet spectrophotometry.The weight coefficient of index components was determined by analytic hierarchy process,and the SAS software was used to optimize the dissolution model and calculate the dissolution parameter T40.MATLAB was used to establish a mathematical model and realize the data visualization between material ratio and dissolution parameter T40.[Results]The mathematical model between material ratio and dissolution parameter was T40=8.93-5.31X1-5.83X2+17.67X1X3(X1,X2,X3 are fiber,starch and grease-resin materials respectively,r2=0.9706,P=0.01,RMSE=0.5696).[Conclusions]Fiber can effectively promote the dissolution(P<0.05).Starch can promote the dissolution,but the effect is not obvious(P=0.05).The interaction between fiber and grease-resin can inhibit the dissolution,but was not significant(P=0.07).

Validation of Microbial Limit Test Methods of Tongmai Tangyanming Capsule

[ Objectives] This study was conducted to establish a method for microbial limit test of Tongmai Tangyanming Capsule. [ Methods] According to the Chinese Pharmacopoei 2015 edition of the four general rules, the microbiological limits of non-sterile products were verified. [ Results] Tongmai Tangyanming Capsule has a strong inhibitory effect on Bacillus subtilis and Candida albicans. The antimicrobial activity was significantly eliminated after increasing the diluent （ 1 ： 20） ; and the recoveries were in the range of 0.5 -2 when the total quantities of aerobic microbes were determined by dilution method （1：20）. When the total quantities of mould and yeast were determined by dilution method （1：20）, the recoveries were from 0.5 to 2, and Escherichia coli, cholate-tolerant Gram-negative bacteria and Salmonella can be detected by the test solution dilution method. [ Condusions] With the plate method, the total quantities of aerobic bacteria and the quantities of mould and yeast were examined by increasing diluent, and conventional method was used for examination of control bacteria including E. coli, cholatetolerant Gram-negative bacteria and Salmonella. This method has been proved effective for microbial limit test of Tongmai Tangyanming Capsule, and can effectively control the quality of the preparation. The method is accurate and reliable.

Effect of Tongmai Huoxue decoction combined with warm needling on hemorheology and antioxidant capacity in patients with high altitude polycythemia

Objective: To investigate the curative effect of Tongmai Huoxue Decoction combined with warm needling on the treatment of High altitude polycythemia (HAPC), and to analyze the changes of hemorheology and antioxidant ability before and after treatment. Methods: A total of 90 HAPC patients were randomly divided into control group and observation group. The control group was treated with low molecular weight dextran and captopril. On the basis of this, the observation group was supplemented with Tongmai Huoxue Decoction combined with warm needling. Hemorheology and antioxidant markers were measured before and after treatment in all subjects. Results: Before treatment, there were no significant differences in the hemorheology indexes between the control group and the observation group. After treatment, the two groups of hemorheology related indicators were significantly lower than those before treatment, including whole blood low shear reduction viscosity, whole blood high shear reduction viscosity, plasma viscosity, Hb, Hct and RBC. The levels of the observation group were significantly lower than that of the control group. Before treatment, there was no significant difference in antioxidant indexes between the control group and the observation group. After treatment, the levels of serum superoxide dismutase (SOD) and glutathione (GSH) in the two groups were higher than those in the pre-treatment, while the content of malondialdehyde (MDA) was lower than that before treatment. The levels of serum SOD and GSH in the observation group were higher than those in the control group, and the MDA content was lower than that in the control group. Conclusion: Routine treatment of Western medicine combined with Tongmai Huoxue Decoction joint warm needling method may reduce hemoglobin, improve hemorheology and improve antioxidant ability in HAPC patients, and it may be one of the potential effective drugs for adjuvant therapy of HAPC.

通脉开窍丸治疗血管性痴呆模型大鼠海马区神经元的铁死亡变化

背景:研究表明铁死亡与血管性痴呆存在密切联系,通脉开窍丸对于改善血管性痴呆患者的认知功能有一定疗效,但其作用机制不明确。目的:基于核因子E2相关因子2(nuclear factor erythroid-2 related factor 2,Nrf2)/血红素氧合酶1(heme oxygenase-1,HO-1)/谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)信号通路调控铁死亡探讨通脉开窍丸对血管性痴呆干预作用以及分子机制。方法:84只雄性SD大鼠,其中12只大鼠用作假手术组,其余大鼠用改良2-VO法制备成血管性痴呆的模型,成模后随机分为模型组、通脉开窍丸高、中、低剂量(27.6,13.8,6.9 g/kg)组、联合组[通脉开窍丸高剂量+ML385(20 mg/kg)]、盐酸多奈哌齐组(0.45 mg/kg),灌胃给药1次/d,联合组同时腹腔注射Nrf2抑制剂ML385,1次/d,连续4周。采用Morris水迷宫检测大鼠的学习记忆能力;苏木精-伊红染色观察各组大鼠海马组织中神经元病理学变化;比色法试剂盒检测大鼠血清中还原型谷胱甘肽、Fe^(2+)、丙二醛的浓度;普鲁士蓝染色法检测大鼠海马组织中铁沉积情况;透射电镜观察大鼠海马组织中神经元线粒体超微结构变化;蛋白免疫印迹法检测大鼠海马神经元Nrf2、HO-1、GPX4、XCT、铁蛋白重链1(ferritin heavy chain 1,FTH1)蛋白的表达。结果与结论:(1)与假手术相比,模型组大鼠逃避潜伏期时间明显延长(P<0.05),穿越平台次数明显减少(P<0.05);海马组织松散,神经元细胞核深染,染色质固缩甚至裂解;CA1区铁离子聚集;线粒体萎缩变小,线粒体嵴溶解消失,线粒体膜密度增厚;血清中Fe^(2+)、丙二醛水平上升,还原型谷胱甘肽水平下降(P<0.05);海马组织GPX4、HO-1、XCT、Nrf2、FTH1蛋白表达显著降低(P<0.05)。(2)与模型组相比,通脉开窍丸各剂量组和盐酸多奈哌齐组大鼠平均逃避潜伏期均明显缩短(P<0.05),穿越平台次数增加(P<0.05);海马神经元恢复明显,CA1区神经元铁离子聚集明显减少,线粒体结构和功能好转;血清Fe^(2+)、丙二醛水平显著降低(P<0.05),血清还原型谷胱甘肽浓度及海马组织中GPX4,HO-1,XCT,Nrf2,FTH1蛋白表达显著升高(P<0.05)。(3)与通脉开窍丸高剂量组相比,盐酸多奈哌齐组治疗效果差异无显著性意义(P>0.05),联合组大鼠水迷宫逃避潜伏期时间延长(P<0.05),穿越平台次数减少(P<0.05),大鼠CA1区神经元病理改变情况不明显,铁沉淀增加,血清中丙二醛、Fe^(2+)浓度增加(P<0.05),还原型谷胱甘肽浓度减少(P<0.05),海马组织神经元线粒体萎缩变小,且Nrf2、XCT、HO-1、GPX4、FTH1蛋白的表达减少(P<0.05)。在一定范围内,通脉开窍丸剂量越高效果越好,且高剂量治疗效果不亚于盐酸多奈哌齐。(4)结果说明,通脉开窍丸可以减轻大鼠海马组织神经元病理改变,改善血管性痴呆大鼠的认知功能,其作用机制可能与Nrf2/HO-1/GPX4信号通路的激活抑制铁死亡有关。

UFLC-DAD-ESI-IT-TOFMSn Analysis on Biotransformation of Tongmai Formula Incubated with Human Intestinal Bacteria

Objective To investigate the biotransformation of Tongmai formula（TMF） in incubated system of human intestinal flora（HIF）. Methods The technique of ultra fast liquid chromatography with diode array detector and coupled with electrospray ionization ion trap time-of-flight multistage mass spectrometry（UFLC-DAD-ESIIT-TOFMSn） was adopted to determine the products of TMF biotransformed by HIF. Results Totally 66 constituents were detected and identified according to the accurate mass measurements（〈 5 ppm） and effective MSn fragment ions. Meanwhile, the potential biotransformational pathways of compounds in TMF transformed by HIF were firstly proposed. Desugarization, hydroxylation, and methylation were the major reactions in the biotransformation mechanism of TMF by HIF. Conclusion This study will be helpful to clarify the material basis of pharmacological activities from TMF in vivo.

Effects of Tongmai Huoxue Yin(通脉活血饮) on Tumor Necrosis Factor-alpha in the Acute Cerebral Ischemia Model Rat

Objective:To observe the interfering action of Tongmai Huoxue Yin(通脉活血饮) on the acute cerebral ischemia model rat.Methods:Total 60 SD rats,30 females and 30 males,were randomly divided into 4 groups,sham-operation group,model group,Nimodipine group and Tongmai Huoxue Yin group,15 rats in each group.The acute cerebral ischemia rat model was duplicated,the middle cerebral artery(MCA) were ligated and the thread was inserted for the rats in the model group,Nimodipine group and Tongmai Huoxue Yin group,for the rats in the sham-operation group,the arteries were separated without ligature and the thread was not inserted.After the modeling has succeed,the water-decocted concentrated solution of 20-fold Tongmai Huoxue Yin clinical dosage was intragastrically administrated in a dose of 3 mL /100 g · d divided into twice,1.5 mL /100 g once.Distilled water 3 mL /100 g·d was intragastrically administrated,1.5 mL /100 g once,for the rat in the model group,Nimodipne suspension 3 mL /100 g·d(0.6 mg /100 g) for the Nimodipine group and 3 mL /100 g · d(5.4g /100 g) for the Tongmai Huoxue Yin group,no drugs for the sham-operation group.And changes of tumor necrosis factor-alpha(TNF-α) contents in the serum and brain tissue were investigated.Results:Compared with the model group,compared with the sham-operation group,serum TNF-α content at 5 h of focal cerebral ischemic ischemia in the model group started to increase and reached to the high peak at 12 h,but in both the Tongmai Huoxue Yin group and the Nimodipine group decreased in varying degrees at the same time;compared with the sham-operation group,brain TNF-α content at 6 h of focal cerebral ischemic ischemia in the model group started to increase and reached to the high peak at 12 h,but in both the Tongmai Huoxue Yin group and the Nimodipine group decreased in varying degrees,with the most obviously decreased at 24 h of ischemia.Tongmai Huoxue Yin could significantly decrease TNF-α content in the brain tissue.Conclusion:Tongmai Huoxue Yin has a protective action on acute cerebral ischemia injury in the rat.

Effects of Bushen Tongmai Recipe(补肾通脉方) on Protein Kinase Bα Expression in Polycystic Ovary Rats with Insulin Resistance

Objective： To observe the effects of Bushen Tongmai Recipe （补肾通脉方, BSTMR） on mRNA and protein expressions of protein kinase B alpha （PKB α） in hepatic, adipose, muscular, and ovarian tissues of polycystic ovary（PCO） rats with insulin resistance （IR） and to explore the possible molecular mechanism of BSTMR in treating IR and ovulation dysfunction. Methods： Female 22-day-old SD rats were injected subcutaneously with sodium prasterone sulfate （9 mg.100g^-1.d^-1） for 20 days and fed with high-fat diet for 80 days to induce PCO rats with IR. Then, the PCO rats were randomly divided into the model group （n=23） and the treated group （n=21）. The treated group was administered with BSTMR for 2 weeks. Meanwhile, a group with 15 rats of the same age was used as the control group. The histological changes in the ovaries were examined. Fasting blood glucose （FBG） was determined by the glucose oxidase method. Serum fasting insulin （Fins） was determined by radioimmunoassay （RIA）. The mRNA level of PKB ~ was measured by reverse transcription polymerase chain reaction （RT-PCR）. Immunohistochemistry staining and Western blot analysis were employed to detect the protein expression in target tissues. Results： Compared with the control group, the ovaries in the model group showed multiple follicular cysts, levels of FBG and Fins in the model group increased markedly （P〈0.05 or P〈0.01, respectively）, and the insulin sensitive index （ISI） decreased obviously （P〈0.01）. The mRNA and protein expressions of PKB e in target tissues in the model group were dramatically lower than those in the control group （P〈0.05 or P〈0.01）. Compared with the model group, the stratum granulosum of the ovarian follicle in the treated group increased markedly, the level of Fins in the treated group decreased obviously （P〈0.01）, ISI in the treated group improved markedly （P〈0.01）, and the mRNA and protein expressions of PKBα in target tissues of the treated rats were elevated significantly （P〈0.05 or P〈0.01）. Conclusion： BSTMR could improve IR and ovulation dysfunction in PCO rats with IR, and its molecular mechanisms might be closely related with the elevation of mRNA and protein expressions of PKB α in target tissues of PCO rats with IR.

Detection of minor constituents in TongMai Granules using NP×RP off-line twodimensional liquid chromatography coupled with orbitrap mass spectrometry

TongMai Granules is a Chinese medicine patent drug to treat cardiovascular diseases.It is prepared from the water extract of Dan-shen,Chuan-xiong and Ge-gen.The main compounds of TongMai Granules are isoflavone glycosides,isoflavones,and phenolic acids.However,it is difficult to detect low-polar compounds such as tanshinones and phthalides though they may also contribute to the therapeutic effect.In this work,an off-line two-dimensional chromatography method was developed to enrich the minor constituents in TongMai Granules.The extract was subjected to an SEP-PAK silica SPE column and eluted with petroleum ether,ethyl acetate–methanol(6:1,v/v),and methanol,successively.The three fractions were then analyzed using ultra-high performance liquid chromatography coupled with orbitrap mass spectrometry(UHPLC/orbitrap-MS).The isoflavones from Ge-gen,phenolic acids from Dan-shen and phthalides from Chuan-xiong were distributed in different fractions.A total of 131 compounds,including 59 from Ge-gen,26 from Dan-shen,43 from Chuan-xiong,and 3 common ones from Dan-shen and Chuan-xiong were tentatively identified.Among them,26 compounds were identified by comparing with reference standards.This study set a good example for comprehensive analysis of the constituents in traditional Chinese medicine formula.

Neuroprotective Effects of Tongmai Yizhi Decoction(通脉益智汤)against Alzheimer's Disease through Attenuating Cyclin-Dependent Kinase-5 Expression

Objectives： To explore the protective effects of Tongmai Yizhi Decoction（通脉益智汤, TYD）, a Chinese herb complex prescription against the impairment of cognitive functions and memory loss in amyloid beta 1–40（Aβ1-40） peptide and ibotenic（IBO）-induced Alzheimer＇s disease（AD） model rats. Methods： The in vivo model was established by injecting Aβ1-40 and IBO into left hippocampal CA1 area of Sprague-Dawley（SD） rat to mimic AD. Totally 32 SD rats were divided into 4 groups, including sham operation group, AD model group, TYD group [AD rats treated with TYD at the dosage of 19.44 g/（kg·d） for 4 weeks] and huperzine A group [AD rats treated with huperzine A at the dosage of 40.5 μg/（kg·d） for 4 weeks]. Spatial learning and memory level was detected by Morris Water Maze test. Histological morphology in the hippocampus was tested by hematoxylin-eosin（HE） staining. Cyclin-dependent kinase-5（Cdk5） protein and gene expression level were investigated by Western blot analysis and real-time quantitative polymerase chain reaction（RT-q PCR）, respectively. Results： Aβ1-40 and IBO treatment induced longer escape latency of rats, compared with sham operation group from day 25（P〈0.01）. However, TYD and huperzine A obviously shortened the escape latency from day 26（P〈0.01）. Moreover, the effect of TYD was similar to huperzine A（P〉0.05）. Furthermore, HE staining also showed that TYD and huperzine A reversed the neuropathological changes in the hippocampus triggered by Aβ1-40 and IBO. TYD and huperzine A effectively reduced the expression levels of Cdk5 protein and gene located in rat hippocampus, compared with the AD model group（P〈0.01）. Conclusion： TYD could be a promising neuroprotective agent for protecting neuron from AD injury through inhibiting Cdk5 expression.

广义估计方程评估调督通脉针灸法治疗不同年龄临床前类风湿关节炎的效果

背景:针灸在类风湿关节炎中的治疗效果尚存在争议。因此,该研究旨在通过广义估计方程评估调督通脉针灸法治疗不同年龄临床前类风湿关节炎患者的效果,为针灸在类风湿关节炎中的应用提供依据。目的:基于广义估计方程探讨调督通脉针灸法对不同年龄临床前类风湿关节炎患者的影响。方法:选取2023年1-9月收治的123例临床前类风湿关节炎患者为研究对象,根据治疗方法不同分为研究组(n=64)和对照组(n=59),研究组给予调督通脉针灸治疗,对照组给予乙酰氨基酚片治疗。采用倾向性评分匹配法调整基线均衡性,比较两组患者临床疗效和治疗前后细胞因子水平,建立广义估计方程模型评估调督通脉针灸法治疗不同年龄临床前类风湿关节炎患者的效果。结果与结论:(1)治疗0 d,研究组和对照组在关节疼痛、C-反应蛋白方面存在显著差异(P<0.05);治疗4周,两组患者在目测类比评分、关节疼痛、C-反应蛋白、红细胞沉降率方面存在显著差异(P<0.05);治疗8周,两组患者在目测类比评分、C-反应蛋白、红细胞沉降率方面存在显著差异(P<0.05);治疗12周,两组患者在目测类比评分、C-反应蛋白、红细胞沉降率方面存在显著差异(P<0.05)。(2)研究组治疗后总有效率为93.75%,对照组为79.17%,研究组患者临床疗效显著优于对照组(P<0.05)。(3)研究组治疗后在白细胞介素6、干扰素γ、巨噬细胞迁移抑制因子、类风湿因子IgA、类风湿因子IgM、基质金属蛋白酶3、基质金属蛋白酶9、抗环状瓜氨酸抗体方面与治疗前相比差异显著(P<0.05);对照组治疗后在白细胞介素6、干扰素γ、类风湿因子IgA、类风湿因子IgM、基质金属蛋白酶3、基质金属蛋白酶9、抗环状瓜氨酸抗体方面与治疗前相比差异显著(P<0.05);研究组治疗后在白细胞介素6、干扰素γ、巨噬细胞迁移抑制因子、类风湿因子IgA、类风湿因子IgM、基质金属蛋白酶3、抗环状瓜氨酸抗体方面与对照组治疗后相比差异显著(P<0.05)。(4)治疗12周,研究组各年龄段患者综合疗效优于对照组(P<0.05);治疗8周,研究组23-35岁、36-50岁、51-60岁患者的综合疗效优于对照组(P<0.05),两组18-22岁患者综合疗效相当;治疗4周,研究组36-50岁、51-60岁患者综合疗效优于对照组(P<0.05),两组18-22岁、23-35岁患者综合疗效相当。结果表明,调督通脉针灸法治疗36-50岁、51-60岁临床前类风湿关节炎患者具有一定优势。

基于消斑通脉方抗动脉粥样硬化作用机制的网络药理学分析和体外实验验证

目的:利用网络药理学分析方法初步预测消斑通脉方抗动脉粥样硬化(AS)的潜在作用通路和靶点,联合体外细胞实验对其可能机制进行验证。方法:采用中药系统药理学数据库与分析平台(TCMSP)、GeneCards、Swiss Target Prediction和Uniprot等数据库,收集消斑通脉方中活性化合物及对应靶点信息,构建“成分-靶点-疾病”网络,通过蛋白-蛋白互作(PPI)网络预测可能的作用靶点和通路,对交集靶点进行基因本体论(GO)功能富集分析和京都基因与基因组百科全书(KEGG)信号通路富集分析。体外培养人主动脉血管平滑肌细胞(HA-VSMCs)并鉴定,采用氧化低密度脂蛋白(ox-LDL)诱导HA-VSMCs异常增殖并进行鉴定。MTT法检测不同浓度消斑通脉方作用后各组HA-VSMCs增殖活性,确定消斑通脉方安全性。HA-VSMCs分为空白组、模型组(诱导HA-VSMCs异常增殖)、瑞舒伐他汀组(诱导HA-VSMCs异常增殖后采用4μmol·L^(−1)瑞舒伐他汀干预)及低、中和高剂量消斑通脉方组(诱导HA-VSMCs异常增殖后分别采用0.025、0.050和0.100 mg·L^(−1)消斑通脉方干预)。酶联免疫吸附试验(ELISA)法检测各组HA-VSMCs培养上清中人单核细胞趋化蛋白1(MCP-1)、白细胞介素6(IL-6)和白细胞介素8(IL-8)水平,实时荧光定量PCR(RT-qPCR)法检测各组HA-VSMCs中核因子κB(NF-κB)p65 mRNA和成纤维细胞生长因子2(FGF2)mRNA表达水平,Western blotting法检测各组HA-VSMCs中NF-κB p65和FGF2蛋白表达水平。结果:消斑通脉方中含有103种活性成分,可通过作用于189个靶基因发挥抗AS作用,潜在作用靶点包括IL-6、IL-8、血管内皮生长因子A(VEGFA)、核因子κB1(NF-κB1)和RELA(NF-κB p65)等。GO功能分析和KEGG信号通路富集分析,消斑通脉方通过调节脂质、缺氧诱导因子1(HIF-1)、表皮生长因子(EGF)、磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)和NF-κB等信号通路发挥抗AS作用。细胞形态表现和免疫荧光染色结果证明细胞为HA-VSMCs。油红O染色,可观察到大量红色脂滴,表明造模成功。MTT法检测,在一定剂量范围内消斑通脉方对HA-VSMCs增殖率无明显影响,安全性良好。ELISA法检测,与模型组比较,瑞舒伐他汀组和不同剂量消斑通脉方组HA-VSMCs培养上清中MCP-1和IL-6水平降低(P<0.05或P<0.01),0.050和0.100 mg·L^(−1)消斑通脉方组HA-VSMC培养上清中IL-8降低(P<0.01);与瑞舒伐他汀组比较,不同剂量消斑通脉方组HA-VSMCs培养上清中MCP-1降低(P<0.01),0.050和0.100 mg·L^(−1)消斑通脉方组HA-VSMCs培养上清中IL-8降低(P<0.01)。与模型组比较,瑞舒伐他汀组和不同剂量消斑通脉方组HA-VSMCs中NF-κB p65 mRNA表达水平降低(P<0.01),瑞舒伐他汀组及0.050和0.100 mg·L^(−1)消斑通脉方组HA-VSMCs中FGF2 mRNA表达水平降低(P<0.01);与瑞舒伐他汀组比较,0.050和0.100 mg·L^(−1)消斑通脉方组HA-VSMCs中NF-κB p65和FGF2 mRNA表达水平降低(P<0.05或P<0.01)。与模型组比较,瑞舒伐他汀组和不同剂量消斑通脉方组HA-VSMCs中NF-κB p65和FGF2蛋白表达水平降低(P<0.01);与瑞舒伐他汀组比较,0.050和0.100 mg·L^(−1)消斑通脉方组HA-VSMCs中NF-κB p65蛋白表达水平降低(P<0.01),0.100 mg·L^(−1)消斑通脉方组HA-VSMCs中FGF2蛋白表达水平降低(P<0.01)。结论:消斑通脉方具有抗炎、抑制HA-VSMCs增殖和抗AS作用,其作用机制可能与NF-κB/FGF2通路失活有关。

基于网络药理学与实验验证探讨通脉颗粒治疗缺血性脑卒中的作用机制

目的采用网络药理学探讨通脉颗粒治疗缺血性脑卒中(ischemic stroke,IS)的作用机制,并开展体内实验进行验证。方法通过TCMSP数据库和TCMIP数据库检索丹参、川芎、葛根3味中药的化学成分及其对应的作用靶点,在GeneCards数据库、Drugbank数据库、TTD数据库、OMIM数据库收集IS的相关靶点;将化学成分靶点和IS靶点绘制韦恩图取交集,借助STRING数据库和Cytoscape软件构建药物-潜在活性成分-作用靶点网络图;利用DAVID数据库对关键靶点进行富集分析。采用大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)法复制IS大鼠模型,造模成功后用通脉颗粒低、中、高剂量药物干预1周。采用Longa评分、免疫荧光染色等方法进行动物体内药效验证和机制探讨。结果经网络药理学分析,共获得通脉颗粒87个潜在活性成分,作用于IS的关键靶点为白细胞介素(interleukin,IL)-6、丝氨酸/苏氨酸蛋白激酶AKT(serine/threonine-protein kinase AKT,AKT)1、肿瘤坏死因子(tumor necrosis factor,TNF)、β-肌动蛋白(beta-actin,ACTB)、血管内皮生长因子(vascular endothelial growth factor,VEGF)A,可能的作用机制与丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)信号通路、磷脂酰肌醇-3-激酶-丝氨酸/苏氨酸蛋白激酶AKT(phosphatidylinositide 3-kinases-serine/threonine-protein kinase AKT,PI3K-Akt)信号通路、TNF信号通路、IL-17信号通路等有关。动物实验验证结果表明,与模型组比较,通脉颗粒可显著降低IS大鼠神经功能缺损评分(P<0.05),能明显缩小脑组织梗死灶、有效改善脑组织病理学表现,改变小胶质细胞活化状态,显著降低IL-6、TNF-α的表达水平(P<0.05),显著升高IL-4和IL-10的表达水平(P<0.05)。结论通脉颗粒可抑制炎症因子的表达,改善神经功能损伤,其作用机制可能与调控小胶质细胞介导的炎症反应有关。

调督通脉针灸法辅助甲氨蝶呤及对乙酰氨基酚治疗类风湿性关节炎患者的疗效观察

目的探究调督通脉针灸法辅助甲氨蝶呤、对乙酰氨基酚在类风湿性关节炎(Rheumatoid arthritis,RA)中的应用效果。方法选取2022年1月—2022年12月期间黑龙江中医药大学附属第四医院RA患者100例,按照随机数字表法分为对照组和研究组,每组各50例。对照组口服甲氨蝶呤,针对疼痛难忍者临时给予对乙酰氨基酚片,研究组在对照组基础上采取调督通脉针灸法。治疗3个月后,观察比较两组患者临床疗效、安全性,治疗前后中医证候积分、疼痛程度(Visual Analogue Scale/Score,VAS)及疾病活动度(Disease Activity Score 28,DAS28)评分、实验室指标[类风湿因子(Rheumatoid factor,RF)、C反应蛋白(C-reactive protein,CRP)、肿瘤坏死因子-α(Tumor necrosis factor-alpha,TNF-α)、抗环瓜氨酸肽抗体(Anti-cyclic citrulline peptide antibody,抗CCP抗体)、血沉(Erythrocyte sedimentation rate,ESR)]水平。结果(1)中医证候积分:治疗后两组患者晨僵、屈伸不利、关节疼痛、关节肿胀积分均较治疗前降低,差异有统计学意义(P<0.05);且研究组低于对照组,差异有统计学意义(P<0.05)。(2)临床疗效:治疗后研究组临床总有效率94.00%(47/50)高于对照组80.00%(40/50),差异有统计学意义(P<0.05)。(3)疼痛程度及疾病活动度:治疗后两组患者VAS、DAS28分值较治疗前降低,差异有统计学意义(P<0.05);且研究组低于对照组,差异有统计学意义(P<0.05)。(4)实验室指标:治疗后两组患者RF、CRP、TNF-α、抗CCP抗体、ESR水平均较治疗前降低,差异有统计学意义(P<0.05);且研究组低于对照组,差异有统计学意义(P<0.05)。(5)安全性:治疗期间,两组患者均未发生血尿常规、肝肾功能异常,且研究组不良反应发生率8.00%(4/50)与对照组4.00%(2/50)比较,差异无统计学意义(P>0.05)。结论采取调督通脉针灸法辅助甲氨蝶呤、对乙酰氨基酚对RA患者实施干预,可减轻患者临床症状,缓解疼痛程度及疾病活动度,调节相关生化指标水平,提升疾病治疗效果,且具有安全性。

基于血小板活化因子的变化分析七叶通脉胶囊及其主要药效物质治疗缺血性脑中风的作用机制

目的:通过观察血小板活化相关因子的变化,分析七叶通脉胶囊(QYTM)治疗缺血性脑中风的作用机制及其药效物质人参皂苷Rb3(Rb3)、三七皂苷R1(R1)、槲皮素的作用特点。方法:采用家兔短暂性全脑缺血再灌注模型和大鼠慢性脑缺血模型,考察了不同剂量QYTM给药后对脑组织中血小板活化因子(PAF)、血小板第四因子(PF4)、β血小板球蛋白(β-TG)及血小板膜糖蛋白Ⅱb/Ⅲa复合物(GPⅡb/Ⅲa)的影响。通过正常小鼠和大鼠脑缺血再灌注模型,考察了QYTM及药效物质槲皮素、R1、Rb3各给药组脑组织中上述因子的变化及对缺血再灌注模型脑梗死面积的影响。结果:QYTM高剂量组能明显降低短暂性全脑缺血再灌注模型家兔脑皮质PAF、PF4、GPⅡb/Ⅲa含量及慢性脑缺血模型大鼠脑组织PF4、GPⅡb/Ⅲa和β-GT的含量(P<0.05,P<0.01);QYTM中剂量组能降低家兔脑皮质PAF、GPⅡb/Ⅲa的含量(P<0.05,P<0.01);QYTM中、低剂量组也能减少大鼠脑组织PF4含量(P<0.01)。QYTM及药效物质槲皮素、R1、Rb3对正常小鼠脑组织中上述因子无明显的影响(P<0.05)。对于大鼠脑缺血再灌注模型,QYTM组脑组织中PAF、PF4、GPⅡb/Ⅲa的含量明显降低(P<0.05,P<0.01),槲皮素组PAF、PF4明显降低(P<0.05,P<0.01);R1组PF4含量降低(P<0.01),Rb3组PAF、PF4、GPⅡb/Ⅲa含量降低(P<0.05,P<0.01),各给药组对脑梗死面积均表现出明显的抑制作用(P<0.05,P<0.01)。结论:QYTM能通过抑制血小板相关因子的活化,发挥活血化瘀作用,进而达到治疗缺血性脑中风的目的。QYTM抑制缺血性脑中风作用与其药效物质皂苷类成分和黄酮类成分的药效作用相关。

论通脉法在慢性心力衰竭治疗中的理论依据与应用

慢性心力衰竭是大部分心血管疾病的最终结局,其5年生存率与恶性肿瘤相当甚至更低。中医学对慢性心力衰竭治疗有多种认识,心病不仅在心,也在血液与血管。致病之“祸”源自血管,血液内的“毒素”长期在血管中运行,逐渐导致血管壁增厚、失去弹性、逐渐硬化,血管通路不畅与心室重塑形成正反馈,不利于慢性心力衰竭的康复。中医学无“血管”之名,只有“脉”。无论气虚、气滞、血瘀、痰浊、寒凝乃至血浊等诸因素,皆可造成脉道痹阻,从而发病。相比传统的强心、养心法,通脉法从整体观的角度,调整气血,通达五脏,有防治慢性心力衰竭的作用。综合运用益气通脉、温阳通脉、理气通脉、化瘀通脉、化浊通脉、利水通脉及解毒通脉等方法,减轻心脏负荷,同时改善血循环,达到开源引流、疏通脉络的目的,以改善慢性心力衰竭的临床症状及延缓病情进展。

养血散寒通脉方治疗血虚寒凝型子宫内膜异位症的临床研究

【目的】观察养血散寒通脉方(由当归四逆加吴茱萸生姜汤加减而成)治疗血虚寒凝型子宫内膜异位症(EMS)的临床疗效。【方法】将120例血虚寒凝型EMS患者随机分为研究组和对照组,每组各60例。研究组给予养血散寒通脉方治疗,对照组给予少腹逐瘀颗粒治疗,疗程为3个月,并于疗程结束后随访1年。观察2组患者治疗前后各种疼痛评分[包括经期腹痛视觉模拟量表(VAS)评分和痛经、非经期盆腔痛、性交痛、盆腔压痛、骶韧带结节触痛分级评分]、卵巢子宫内膜异位囊肿大小及血清糖类抗原125(CA125)、血管内皮生长因子(VEGF)、可溶性细胞间黏附分子1(SICAM-1)、基质金属蛋白酶9(MMP-9)、组织金属蛋白酶抑制因子2(TIMP-2)水平的变化情况,并观察2组患者的临床疗效、安全性、复发情况及妊娠情况。【结果】(1)研究过程中,研究组脱落3例,对照组脱落5例,最终共112例患者纳入统计分析,其中研究组57例,对照组55例。(2)治疗3个月后,研究组的总有效率为92.98%(53/57),对照组为85.45%(47/55),组间比较,研究组的疗效明显优于对照组(P<0.05)。(3)治疗后,2组患者的各种疼痛评分(包括经期腹痛VAS评分和痛经、非经期盆腔痛、性交痛、盆腔压痛、骶韧带结节触痛分级评分)均较治疗前明显下降(P<0.05),且研究组的下降幅度均明显优于对照组(P<0.05)。(4)治疗后,2组患者的卵巢子宫内膜异位囊肿均略有缩小,但组内治疗前后及治疗后组间比较,差异均无统计学意义(P>0.05)。(5)治疗后,2组患者血清CA125、VEGF、SICAM-1、MMP-9水平均较治疗前下降(P<0.05),血清TIMP-2水平均较治疗前升高(P<0.05),且研究组对血清CA125、VEGF、SICAM-1、MMP-9水平的下降幅度及对血清TIMP-2水平的升高幅度均明显优于对照组(P<0.05)。(6)随访1年,研究组的复发率为30.19%(16/53),明显低于对照组的68.09%(32/47),组间比较,差异有统计学意义(P<0.05)。研究组21例有生育要求,其中14例妊娠,妊娠率为66.67%(14/21);对照组20例有生育要求,其中4例妊娠,妊娠率为20.00%(4/20);组间比较,研究组的妊娠率明显高于对照组,差异有统计学意义(P<0.05)。(7)治疗期间,2组患者均无明显不良反应发生,且患者的血、尿、大便常规及心电图、肝肾功能等安全性指标均无异常变化。【结论】养血散寒通脉方治疗血虚寒凝型EMS患者疗效确切,能够显著缓解患者各种疼痛症状,改善妊娠结局,有效调节血清CA125、VEGF、SICAM-1、MMP-9、TIMP-2水平。