Neuroprotective effects of Longxue Tongluo Capsule on ischemic stroke rats revealed by LC-MS/MS-based metabolomics approach / 中草药·英文版

OBJECTIVE@#The present study aimed to evaluate the therapeutic effect and explore the underlying mechanisms of Longxue Tongluo Capsule (LTC) on ischemic stroke rats.@*METHODS@#Twenty-six rats were randomly divided into four groups, including sham group, sham + LTC group, MCAO group, and MCAO + LTC group. Ischemic stroke rats were simulated by middle cerebral artery occlusion (MCAO), and LTC treatment group were orally administrated with 300 mg/kg of LTC once daily for seven consecutive days. LTC therapy was validated in terms of neurobehavioral abnormality evaluation, cerebral infarct area, and histological assessments. The plasma metabolome comparisons amongst different groups were conducted by UHPLC-Q Exactive MS in combination with subsequent multivariate statistical analysis, aiming to finding the molecules in respond to the surgery or LTC treatment.@*RESULTS@#Intragastric administration of LTC significantly decreased not only the neurobehavioral abnormality scores but also the cerebral infarct area of MCAO rats. The interstitial edema, atrophy, and pyknosis of glial and neuronal cells occurred in the infarcted area, core area, and marginal area of cerebral cortex were improved after LTC treatment. A total of 13 potential biomarkers were observed, and Youden index of 11 biomarkers such as LysoPC, SM, and PE were more than 0.7, which were involved in neuroprotective process. The correlation and pathway analysis showed that LTC was beneficial to ischemic stroke rats via regulating glycerophospholipid and sphingolipid metabolism, together with nicotinate and nicotinamide metabolism. Heatmap and ternary analysis indicated the synergistic effect of carbohydrates and lipids may be induced by flavonoid intake from LTC.@*CONCLUSION@#The present study could provide evidence that metabolomics, as systematic approach, revealed its capacity to evaluate the holistic efficacy of TCM, and investigate the molecular mechanism underlying the clinical treatment of LTC on ischemic stroke.

Protective Effect of Longxue Tongluo Capsule on H2O2-induced Injury of PC12 Cells / 中国实验方剂学杂志

Objective::To investigate the protective effect of Longxue Tongluo capsule (LTC) on H2O2-induced injury of PC12 cells and its potential mechanism. Method::An in vitro injury model in PC12 cells was established with 500 μmol·L-1 H2O2.The experiment was divided into control group, injury model group (H2O2 500 μmol·L-1), and Longxuetongluo capsule group (LTC, 1, 2, 4 mg·L-1). Cell counting kit-8 (CCK-8) assay, intracellular reactive oxygen species (ROS), mitochondrial membrane potential, apoptosis of PC12 cells, and Western blot were used to evaluate the protective effect of LTC on PC12 cells induced by H2O2. Result::Compared with the control group, the cell viability was significantly decreased in the injury model group (P<0.01), intracellular ROS level was significantly increased (P<0.01), mitochondrial membrane potential was decreased, while apoptosis of PC12 cells was significantly increased (P<0.01), and the expression of cleaved poly adenosine diphospho ribose polymerase (PARP) was also increased significantly (P<0.01). Compared with the injury model group, pretreatment with LTC at the concentrations of 2 and 4 mg·L for 6 h significantly increased cell viability in PC12 cells exposed to H2O2 (P<0.01). Moreover, pretreatment with LTC reduced intracellular ROS level (P<0.05), maintained mitochondrial membrane potential, and inhibited apoptosis of PC12 cells induced by H2O2 in a dose-dependent manner (P<0.01). The results of western blotting showed that pretreatment with LTC significantly reduced the expression of cleaved PARP (P<0.01). Conclusion::LTC exerts a significant protective effect against H2O2-induced PC12 cells injury through inhibition of neuronal apoptosis by suppressing intracellular oxidative stress, maintaining mitochondrial function, and promoting DNA repair.

Protective effect of Longxue Tongluo capsule on oxidized low-density lipoprotein damage to human umbilical vein endothelial cells / 中国中药杂志

To observe the protective effect of Longxue Tongluo capsule (LTC) on human umbilical vein endothelial cells (EAhy.926 cells) injury induced by oxidized low-density lipoprotein (ox-LDL, 100 mg·L⁻¹). The effect of the cell viability of LTCin alleviating OX-LDL-induced endothelial cell injury was determined by MTT and LDH assay. The effect of LTC on lactic dehydrogenase (LDH), nitric oxide (NO), super oxide dlsmutase (SOD) and malondialdehyde (MDA) levels were detected by corresponding assay kits according to manufacturer's instruction. The effect of LTC on the protein expressions of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), p65, p-p65, IKB and p-IKB were detected by Western blot. The results showed that compared with the normal control group, the activity of EAhy.926 cells was significantly decreased, LDH leakage (<0.01) increased, NO content and SOD activity significantly decreased (<0.01, <0.05), and the expressions of ICAM-1, VCAM-1, p-p65/p65 and p-IKB(<0.05)increased.This study demonstrated that LTC had no significant effect on the growth of normal cells. The treatment with LTC significantly promoted the proliferation of vascular endothelial cells damagedby ox-LDL, decreased MDA content and LDH release, andincreased the activity of SOD and NO content. Meanwhile, ox-LDL significantly increased the expressions of ICAM-1, VCAM-1, p-p65/p65, p-IKB/IKB in Eahy.926 cells; these effects were suppressed by LTC at 1, 2 mg·L⁻¹. In conclusion, LTC has a significant protective effect on human umbilical vein endothelial cells caused by ox-LDL. This study suggested that LTC has a certain therapeutic effect on AS.

A multi-center, double-blind, placebo-controlled, randomized trial for effect of Longxue Tongluo capsule in treatment of patients of atherosclerotic thrombotic cerebral infarction with blood-stasis syndrome in convalescence / 中国中药杂志

To evaluate the effectiveness and safety of Longxue Tongluo capsule on patients of atherosclerotic thrombotic cerebral infarction convalescence with blood-stasis syndrome, a double-blind, randomized controlled, multi-center clinical trial was conducted. A total of 160 eligible patients were randomly divided into treatment group and control group, with 80 patients in each group, and all of them were orally given Troxerutin pill(three pills each time, three times daily). Longxue Tongluo capsule was applied in the treatment group, while placebo was applied in the control group(two capsules each time, three times daily) for 4 weeks. Main outcomes were measured by ITT analysis. The neurological function deficits scale showed a decrease of 5.17±2.60 in the treatment group, while 4.31±2.31 in the control group, with significant differences between the two groups(P<0.05); the reduction rate in the treatment group (37.2±15.8)% was significantly higher than that in the control group (29.9±15.3)%(P<0.05). In terms of the comprehensive curative effect by ITT, the effective rates in the treatment and control group were 31.6% and 13.5%, respectively(P<0.05). With respect to the efficacy of traditional Chinese medicine syndrome by ITT, the total effective rate of the treatment group was significantly higher than the control group 88.2% vs 68.9%, P<0.05. Three cases of adverse events occurred in this study, including 1 case of diarrhea in treatment group and 2 cases of skin itch and upper respiratory infection in control group. In conclusion, Longxue Tongluo capsule is effective and safe in the treatment of patients of atherosclerotic thrombotic cerebral infarction convalescence with blood-stasis syndrome, and can effectively alleviate the patients' nerve function defect degree and invalidism, with a good effect on blood stasis syndrome.