Protective effects of GuanXinNing tablet (GXNT) on diabetic encephalopathy in zucker diabetic obesity (ZDF) rats.

BACKGROUND: Diabetic encephalopathy (DE) is a complication of diabetes that leads to cognitive and behavioral decline. Utilizing safe and effective complementary and alternative medications for its management is a wise choice. Previous studies have shown that GuanXinNing Tablet (GXNT), an oral preparation primarily derived from two Chinese herbs, Salvia miltiorrhiza Bge. and Ligusticum chuanxiong Hort., exerts a beneficial neuroprotective effect. In this study, we explored the protective effects of GXNT on DE in male Zucker diabetic fatty (ZDF) rats induced by a high-fat diet, aiming to ascertain its significance and potential mechanisms. METHODS: ZDF rats were induced to develop type 2 diabetes (T2DM) with DE by a high-fat diet and treated with GXNT for 8 weeks until they were 20 weeks old. Throughout the experiment, the animals' vital parameters, such as body weight, were continuously monitored. Cognitive function was evaluated using the Y maze test. Biochemical kits were employed to analyze blood glucose, lipids, and vascular endothelial-related factors. Cerebrovascular lesions were assessed using magnetic resonance angiography (MRA) imaging. Brain lesions were evaluated using hematoxylin and eosin (H&E) staining and ultrastructure observation. IgG and albumin (ALB) leakage were detected using immunofluorescence. RESULTS: GXNT demonstrated an enhancement in the overall well-being of the animals. It notably improved cognitive and behavioral abilities, as demonstrated by extended retention time in the novel heterogeneous arm during the Y-maze test. GXNT effectively regulated glucose and lipid metabolism, reducing fasting and postprandial blood glucose, glycated hemoglobin (HbA1c), and total cholesterol (TC) levels. Additionally, it exhibited a protective effect on the vascular endothelium by reducing the serum TXB2/PGI2 ratio while elevating NO and PGI2 levels. Moreover, GXNT ameliorated stenosis and occlusion in cerebral vessel branches, increased the number of microvessels and neurons around the hippocampus, and improved microvascular occlusion in the cerebral cortex, along with addressing perivascular cell abnormalities. Immunofluorescence staining showed a decrease in the fluorescence intensity of IgG and ALB in the cerebral cortex. CONCLUSIONS: GXNT demonstrated a highly satisfactory protective effect on DE in ZDF rats. Its mechanism of action could be based on the regulation of glucolipid metabolism and its protective effect on the vascular endothelium.

Guanxinning tablets improve myocardial hypertrophy by inhibiting the activation of MEK-ERK1/2 signaling pathway.

Myocardial hypertrophy may lead to heart failure and sudden death. As traditional Chinese medicine, Guanxinning tablets (GXN) have significant pharmacological effects in the prevention and treatment of cardiovascular diseases. However, the anti-cardiac hypertrophy efficacy of GXN and its mechanism of action are still unclear. Therefore, we established a heart failure rat model and isolated primary cardiomyocytes of neonatal rat to observe the protective effect of GXN on heart failure rat model and the intervention effect on myocardial cell hypertrophy, and to explore the possible mechanism of GXN preventing and treating myocardial hypertrophy. The results of in vivo experiments showed that GXN could significantly reduce the degree of cardiac hypertrophy, reduce the size of cardiomyocytes, inhibit the degree of myocardial remodeling and fibrosis, and improve cardiac function in rats with early heart failure. The results of in vitro experiments showed that GXN was safe for primary cardiomyocytes and could improve cardiomyocyte hypertrophy and reduce the apoptosis of cardiomyocytes in pathological state, which may be related to the inhibition of the over-activation of MEK-ERK1/2 signaling pathway. In conclusion, GXN may inhibit cardiac hypertrophy and improve early heart failure by inhibiting the over-activation of MEK-ERK1/2 signaling pathway.

Guanxinning Tablet and their active substances alleviate ox-LDL-induced endothelial cell injury / 药学学报

This study investigated the intervention effect of Guanxinning Tablet on human umbilical vein endothelial cells (HUVECs) injury induced by oxidized low density lipoprotein (ox-LDL), providing experimental basis for Guanxinning Tablet in the treatment of atherosclerosis-related diseases. Under the damage of HUVECs by ox-LDL, the cell viability was detected by CCK-8 (cell counting kit-8) assay; lactate dehydrogenase (LDH) in the cell culture supernatant was detected by the corresponding kit; the cell morphology of different groups was observed by common phase contrast microscope; reactive oxygen species (ROS) and NO levels in the cells were detected by DCFH-DA and DAF-FM DA probes, respectively; monocyte adhesion assay was used to detect the recruitment of THP-1 in HUVECs, and TMRM dye was used to detect the level of mitochondrial membrane potential; interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) secretion in the cells was detected by ELISA assay. The results showed that Guanxinning Tablet had a concentration-dependent proliferative effect on HUVECs. Under the stimulation of 100 μg·mL-1 ox-LDL, the morphology of endothelial cells was significantly changed. At this time, NO level was significantly decreased, ROS level was significantly increased and accompanied by a decrease in mitochondrial membrane potential. The recruitment of THP-1 cells by endothelial cells and IL-6, ICAM-1 and MCP-1 were also significantly increased, resulting in oxidative stress and inflammatory injury. Guanxinning Tablet and its composed extracts could significantly improve cell morphology, increase NO level, decrease ROS production, and also reduce the secretion of inflammation-related proteins IL-6 and MCP-1. Salvia miltiorrhiza and Ligusticum striatum DC. have significant synergistic effects on NO. Among them, salvianolic acid B and salvianic acid A exerted the main effects, and the combined efficacy of salvianic acid A and ferulic acid was superior to that of single administration. The above results showed that Guanxinning Tablet and their active substances had the effects of improving endothelial basal function, resisting oxidative stress, and alleviating inflammatory injury, and Salvia miltiorrhiza and Ligusticum striatum DC. synergized, which may be related to their regulation of oxidative stress and inflammation and have application prospects in the treatment of atherosclerosis-related diseases.

Guanxinning tablet inhibits the interaction between leukocyte integrin Mac-1 and platelet GPIbα for antithrombosis without increased bleeding risk.

Recent studies have showed that thrombosis is closely related to leucocytes involved in immunity. Interfering with the binding of leukocyte integrin Mac-1 and platelet GPIbα can inhibit thrombosis without affecting physiological coagulation. Mac-1-GPIbα is proposed as a potential safety target for antithrombotic agents. Guanxinning tablet (GXNT) is an oral Chinese patent medicine used for the treatment of angina pectoris, which contains phenolic acid active ingredients, such as salvianolic acids, ferulic acid, chlorogenic acid, caffeic acid, rosmarinic acid, tanshinol, and protocatechualdehyde. Our previous studies demonstrated that GXN exhibited significant antithrombotic effects, and clinical studies suggested that it did not increase bleeding risk. In addition, GXN exerted a significantly regulatory effect on immune inflammation. In the current study, we intended to evaluate the effects of GXN on bleeding events and explore the safety antithrombotic mechanism of GXN based on leukocyte-platelet interaction. First, we established a gastric ulcer model induced by acetic acid in rats and found that GXN not only did not increase the degree of gastrointestinal bleeding when gastric ulcer occurred, but also had a certain promoting effect on the healing of gastric ulcer. Second, in vitroexperiments showed that after pretreatment with GXN and activation by phorbol 12-myristate-13-acetate (PMA), the adhesion and aggregation of leukocytes with human platelets were reduced. It was also found that GXN reduced the expression and activation of Mac-1 in leucocytes, and inhibited platelet activation due to leukocyte engagement via Mac-1. Overall, the results suggest that GXN may be a safe antithrombotic agent, and its low bleeding risk mechanism is probably related to inhibited leukocyte-platelet aggregation and its interaction target Mac-1-GPIbα.

Vasodilatory Effect of Guanxinning Tablet on Rabbit Thoracic Aorta is Modulated by Both Endothelium-Dependent and -Independent Mechanism.

Vasodilatory therapy plays an important role in the treatment of cardiovascular diseases, especially hypertension and coronary heart disease. Previous research found that Guanxinning tablet (GXNT), a traditional Chinese compound preparation composed of Salvia miltiorrhiza (Danshen) and Ligusticum chuanxiong (Chuanxiong), increase blood flow in the arteries, but whether vasodilation plays a role in this effect remains unclear. Here, we found that GXNT significantly alleviated the vasoconstriction of isolated rabbit thoracic aorta induced by phenylephrine (PE), norepinephrine (NE), and KCl in a dose-dependent manner with or without endothelial cells (ECs). Changes in calcium ion levels in vascular smooth muscle cells (VSMCs) showed that both intracellular calcium release and extracellular calcium influx through receptor-dependent calcium channel (ROC) declined with GXNT treatment. Experiments to examine potassium channels suggested that endothelium-denuded vessels were also regulated by calcium-activated potassium channels (Kca) and ATP-related potassium channels (KATP) but not voltage-gated potassium channels (kv) and inward rectifying potassium channels (KIR). For endothelium-intact vessels, the nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) contents in vascular tissue obviously increased after GXNT treatment, and pretreatment with the NO synthase inhibitor Nw-nitro-L-arginine methyl ester (L-NAME) or guanylyl cyclase inhibitor methylthionine chloride (MB) significantly inhibited vasodilation. An assessment of NO-related pathway protein expression revealed that GXNT enhanced the expression of phosphorylated endothelial NO synthase (eNOS) in a dose-dependent manner but had no effect on total eNOS, p-Akt, Akt, or PI3K levels in human umbilical vein ECs (HUVECs). In addition to PI3K/AKT signaling, Ca2+/calmodulin (CaM)-Ca2+/CaM-dependent protein kinase II (CaMKII) signaling is a major signal transduction pathway involved in eNOS activation in ECs. Further results showed that free calcium ion levels were decreased in HUVECs with GXNT treatment, accompanied by an increase in p-CaMKII expression, implying an increase in the Ca2+/CaM-Ca2+/CaMKII cascade. Taken together, these findings suggest that the GXNT may have exerted their vasodilative effect by activating the endothelial CaMKII/eNOS signaling pathway in endothelium-intact rings and calcium-related ion channels in endothelium-denuded vessels.

Network Pharmacology-Based Approach Uncovers the Mechanism of GuanXinNing Tablet for Treating Thrombus by MAPKs Signal Pathway.

BACKGROUND: GuanXinNing tablet (GXNT), a traditional Chinese patent medicine, has been found to have remarkable antithrombotic effects and can effectively inhibit pro-thrombotic factors in previous studies. However, the mechanism of its antithrombotic effects remains little known. METHODS: In this study, we first determined and identified the sources of each main compound in GXNT using liquid chromatography-mass spectrometry (LC-MS). Through the approach of network pharmacology, we predicted the action targets of the active components, mapped the target genes related to thrombus, and obtained potential antithrombotic targets for active ingredients. We then performed gene ontology (GO) enrichment analyses and KEGG signaling pathway analyses for the action targets, and constructed networks of active component-target and active component-target-pathway for GXNT. Additionally, we evaluated the pharmacodynamic effects of GXNT on thrombus using the rat thrombus model induced by FeCl3, observed the effects of antiplatelet aggregation via platelet assay, and further verified the results predicted by network pharmacology via Western blot. RESULTS: In total, 14 active ingredients were identified in GXNT, and 83 action targets were predicted, 17 of which are antithrombotic targets that potentially participate in processes including response to oxidative stress and positive regulation of blood vessel endothelial cell migration. KEGG pathway analyses revealed that the predicted action targets were involved in multiple signal pathways, such as MAPK, IL-17, and platelet activation. Pharmacodynamics study found that GXNT could significantly reduce the thrombus length and weight, lower platelet aggregation function, and decrease the levels of Fbg and PAI-1. In addition, GXNT could significantly increase 6-keto-PGF1α content and regulate the ratio of TXB2/6-keto-PGF1α, while not having dramatic effects on TXB2. GXNT was also observed to visibly inhibit maximum platelet aggregation. Herein, we further studied the thrombus-related MAPKs signaling pathway and found that GXNT could significantly reduce the phosphorylation levels of p38MAPK, ERK, and JNK proteins in platelet. CONCLUSIONS: This study revealed the pharmacodynamic material basis of GXNT and its potential multicomponent-multitarget-multipath pharmacological effects, confirmed the antithrombotic effects of GXNT, and showed that its mechanism may be related to inhibiting phosphorylation of p38, ERK, and JNK proteins in MAPKs signaling pathway, partially verifying the results from network pharmacology. The results from this study could provide a theoretical basis for the development and clinical application of GXNT.

Effect and Safety of Guanxinning Tablet () for Stable Angina Pectoris Patients with Xin (Heart)-Blood Stagnation Syndrome: A Randomized, Multicenter, Placebo-Controlled Trial.

OBJECTIVE: To investigate the effect and safety of Guanxinning Tablet (, GXN) for the treatment of stable angina pectoris patients with Xin (Heart)-blood stagnation syndrome (XBSS). METHODS: One hundred and sixty stable angina pectoris patients with XBSS were randomly assigned to receive GXN (80 cases) or placebo (80 cases, Guanxinning simulation tablets, mainly composed of lactose), 4 tablets (0.38 g/tablet), thrice daily for 12 weeks. After treatment, an exercise stress test (treadmill protocol), Chinese medicine (CM) syndrome score, electrocardiogram (ECG), and nitroglycerin withdrawal rate were evaluated and compared in the patients between the two groups. Meanwhile, adverse events (AEs) were evaluated during the whole clinical trial. RESULTS: Compared with the control group, the time extension of exercise duration in the GXN group increased 29.28 ±17.67 s after treatment (P>0.05); moreover, the change of exercise duration in the GXN group increased 63.10 ±96.96 s in subgroup analysis (P<0.05). The effective rates of angina pectoris, CM syndrome and ECG as well as nitroglycerin withdrawal rate were 81.33%, 90.67%, 45.76%, and 70.73%, respectively in the GXN group, which were all significantly higher than those in the control group (40.58%, 75.36%, 26.92%, 28.21%, respectively, P<0.05). CONCLUSION: GXN was a safe and effective treatment for stable angina pectoris patients with XBSS at a dose of 4 tablets, thrice daily.

Effect and Safety of Guanxinning Tablet () for Stable Angina Pectoris Patients with Xin (Heart)-Blood Stagnation Syndrome: A Randomized, Multicenter, Placebo-Controlled Trial / 中国结合医学杂志

OBJECTIVE@#To investigate the effect and safety of Guanxinning Tablet (, GXN) for the treatment of stable angina pectoris patients with Xin (Heart)-blood stagnation syndrome (XBSS).@*METHODS@#One hundred and sixty stable angina pectoris patients with XBSS were randomly assigned to receive GXN (80 cases) or placebo (80 cases, Guanxinning simulation tablets, mainly composed of lactose), 4 tablets (0.38 g/tablet), thrice daily for 12 weeks. After treatment, an exercise stress test (treadmill protocol), Chinese medicine (CM) syndrome score, electrocardiogram (ECG), and nitroglycerin withdrawal rate were evaluated and compared in the patients between the two groups. Meanwhile, adverse events (AEs) were evaluated during the whole clinical trial.@*RESULTS@#Compared with the control group, the time extension of exercise duration in the GXN group increased 29.28 ±17.67 s after treatment (P>0.05); moreover, the change of exercise duration in the GXN group increased 63.10 ±96.96 s in subgroup analysis (P<0.05). The effective rates of angina pectoris, CM syndrome and ECG as well as nitroglycerin withdrawal rate were 81.33%, 90.67%, 45.76%, and 70.73%, respectively in the GXN group, which were all significantly higher than those in the control group (40.58%, 75.36%, 26.92%, 28.21%, respectively, P<0.05).@*CONCLUSION@#GXN was a safe and effective treatment for stable angina pectoris patients with XBSS at a dose of 4 tablets, thrice daily.

Guanxinning tablet for patients who switch from dual antiplatelet therapy to aspirin alone after percutaneous coronary intervention: study protocol for a cluster randomized controlled trial.

BACKGROUND: One-year dual antiplatelet therapy (DAPT), generally aspirin in combination with a P2Y12 receptor inhibitor, has been a standard treatment for patients undergoing percutaneous coronary intervention (PCI). Prolonged DAPT has proven itself effective in further reducing cardiovascular events, yet with increased risk of bleeding. Thus, it is of great necessity to find an alternative drug that is as effective but safer and more economic than the P2Y12 inhibitors after termination of one-year DAPT. METHODS: We will conduct a cluster randomized controlled trial in 3600 eligible post-PCI patients from 36 tertiary hospitals (100 patients per hospital) across mainland China. The hospitals served as clusters are randomized in a 2:1 ratio to Guanxinning tablet (GXNT) plus aspirin or aspirin alone for 12 months, with other conventional treatment applied in both groups. After the treatment period, all patients will be followed up for another 12 months. The primary outcome measure is composite cardiovascular events including cardiovascular death, non-fatal myocardial infarction, stent thrombosis, revascularization, ischemic stroke, and re-admission due to unstable angina. Secondary outcome measures are all-cause mortality, each individual component of the primary outcome measure, and stopping or reducing the rate of nitroglycerin administration. Adverse events, including bleeding, will be closely monitored during the whole trial period. In addition, a cost-effectiveness study of GXNT for the study population will be conducted along with this trial. DISCUSSION: This trial aims to determine whether the addition of GXNT will further improve prognosis without increasing bleeding risk for patients with coronary artery disease who have switched from DAPT to aspirin alone after PCI. Completion of this clinical trial might provide a novel, promising, and safer alternative to P2Y12 inhibitors for prolonged antiplatelet therapy in post-PCI patients. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR-IIR-17010688 . Registered on 20 February 2017.

Study on the protective effect of Guanxinning Tablet on myocardial ischemia reperfusion injury in rats / 中国比较医学杂志

Objective To observe the effect of Guanxinning Tablet (GXNT) on myocardial infarction and cardiac autonomic nervous function in rats with myocardial ischemia reperfusion injury (MI/RI).Methods Seventy SD rats were divided into 7 groups randomly (n=10);the sham group, the MI/RI group, 75 mg/kg, 150 mg/kg, 300 mg/kg and 600 mg/kg GXNT groups and 300 mg/kg Compound Danshen Tablets (DST) group.All rats were administered orally for 7 days, and then the MI/RI model was made by ligating the left anterior descending branch of coronary artery in rats.The changes of electrocardiogram were recorded and the electrocardiogram of J points and heart rate variability (HRV) parameters were analyzed.At the end of reperfusion, the myocardial infarct size was measured by using Evans blue and tetrazolium chloride (TTC) double staining, and pathological changes of myocardium were observed by HE staining.The changes of serum lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA) and nitric oxide (NO) levels were also detected.Results Compared with MI/RI group, GXNT and DST groups were significantly reduced myocardial infarct size and inhibited the rising of serum LDH and CK activities (P < 0.05, P < 0.01), and also reduced the total or average value of J point during reperfusion (P < 0.05, P < 0.01).Meanwhile, GXNT and DST groups were markedly increased HRV and serum NO level as well as decreased serum MDA content (P < 0.05, P < 0.01), and improved myocardial tissue pathology.Conclusions GXNT can reduce the myocardial infarction in rats with MI/RI, and also improve the cardiac autonomic nervous function.

Establishment of a zebrafish model of thrombosis and the intervention effect of Guanxinning tablet / 中国实验动物学报

Objective To establish a zebrafish model of thrombosis induced by three kinds of inducers and observe the anti?thrombotic effect of a Chinese traditional medicine, Guanxinning tablet ( GXN) . Methods The zebrafish models of thrombosis was induced by using 1?5μmol/L phenyl hydrazine, 80μmol/L arachidonic acid and 5 mg/L ponatinib, re?spectively, and were treated with various concentration of GXN, clopidogrel or asprin. The thrombus in the tail vein was observed under microscope, Erythrocytes in the zebrafish heart were stained with o?dianisidine and the erythrocyte staining intensity was assessed with a NIS?Elements DTM image analyzer, and the anti?thrombolic effect of GXN was calculated. Results Venous thrombus was significantly increased and the staining intensities of erythrocytes in the heart were signifi?cantly decreased after induction by phenyl hydrazine, arachidonic acid or Ponatinib ( P <0?001 ) , respectively. At the same time, GXN showed an incresing anti?thrombolic effect in the zebrafish models (P<0?001) in a dose?effect manner, with a IC50 of GXN of 44?32 mg/L,138?5 mg/L and 459?5 mg/L, respectively. Conclusions The zebrafish models of thrombosis are successfully established by phenyl hydrazine, arachidonic acid or Ponatinib, respectively, by different for?mation mechanisms. GXN has been shown to have an anti?thrombosis effect, probably, by multiple target effects.