Longxuetongluo Capsule Research Articles

Neuroprotective effects of Longxue Tongluo Capsule on ischemic stroke rats revealed by LC-MS/MS-based metabolomics approach

ObjectiveThe present study aimed to evaluate the therapeutic effect and explore the underlying mechanisms of Longxue Tongluo Capsule (LTC) on ischemic stroke rats. MethodsTwenty-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. ResultsIntragastric 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. ConclusionThe 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.

Longxuetongluo Capsule protects against cerebral ischemia/reperfusion injury through endoplasmic reticulum stress and MAPK-mediated mechanisms

IntroductionLongxuetongluo Capsule (LTC) is wildly applied to treat ischemic stroke in clinical practice in China. However, the pharmacological mechanism of LTC on ischemic stroke is still unstated. ObjectiveOur research was designed to study the protective effect of LTC against cerebral ischemia–reperfusion (I/R) injury and reveal the underlying mechanism both in vivo and in vitro. MethodsPC12 cells treated with glucose deprivation/reperfusion (OGD/R) were used to simulate in vitro ischemia/reperfusion (I/R) injury. The cell viability, apoptosis rate, and protein expressions of PC12 cells were evaluated. In vivo validation of the protective effect of LTC was carried out by middle cerebral artery occlusion (MCAO)/reperfusion treatment, and the underlying mechanism of its anti-apoptosis ability was further revealed by immunohistochemistry staining and Western blotting. ResultsIn the current study, we observed that LTC effectively inhibited oxygen-glucose deprivation/reperfusion (OGD/R) induced apoptosis of PC12 cells through suppressing the cleavage of poly ADP-ribose polymerase (PARP), caspase-3, and caspase-9. Further investigation revealed that OGD/R insult remarkably triggered the endoplasmic reticulum stress responses (ER stress) to induce PC12 cell apoptosis. LTC treatment alleviated OGD/R induced ER stress by inhibiting the activation of protein kinase RNA (PKR)-like ER kinase (PERK)/eukaryotic translation initiation factor 2 (eIF2α) and inositol requiring enzyme 1 (IRE1)/tumor necrosis factor receptor-associated factor 2 (TRAF2) pathways. Additionally, LTC also restrained the OGD/R-induced PC12 cell apoptosis by reversing the activated mitogen-activated protein kinase (MAPK) through IRE1/TRAF2 pathway. Animal studies demonstrated LTC significantly restricted the infarct region induced by middle cerebral artery occlusion (MCAO)/reperfusion, the activation of ER stress and apoptosis of neuronal cells had also been suppressed by LTC in the penumbra region. ConclusionLTC protects the cerebral neuronal cell against ischemia/reperfusion injury through ER stress and MAPK-mediated mechanisms.

Neuroprotective Effects of Longxuetongluo Capsule on Ischemic Stroke Rats Revealed by LC-MS/MS-Based Metabolomics Approach

Longxuetongluo Capsule (LTC) that was prepared from the phenolic compound cluster of Chinese dragon’s blood, has been clinically utilized for years towards ischemic stroke. However, LTC therapeutic mechanisms remain unclear, which restricts its further application and promotion. The present study aimed to evaluate the therapeutic effect and to explore the underlying mechanisms of LTC on ischemic stroke rats characterized by cerebral ischemia reperfusion injury. Ischemic stroke rats were simulated by middle cerebral artery occlusion (MCAO), and LTC therapy was validated by the assays in terms of histological assessments. The plasma metabolite profiling was used to clarify the molecules in respond to the LTC treatment. A total of 13 potential biomarkers contributing to ischemic stroke were observed, and Youden index of 11 biomarkers such as LysoPC, SM, and PE (24:1/18:3) were more than 0.7, which are 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 bicotinate and nicotinamide metabolism. Heatmap and ternary analysis indicated the synergistic effect of carbohydrates and lipids may be induced by flavonoid intake from LTC. Funding Information: This work was financially supported by the National Natural Science Foundation of China (81573572, 81530097), China Agriculture Research System of MOF and MARA, and Central Public-interest Scientific Institution Basal Research Fund (Y2020XK24) Declaration of Interests: Authors declare no conflict of interest. Ethics Approval Statement: All animal experiment protocols were approved by the Medical Ethics Committee of Beijing University of Chinese Medicine

Longxuetongluo capsule alleviates lipopolysaccharide-induced neuroinflammation by regulating multiple signaling pathways in BV2 microglia cells.

Longxuetongluo capsule (LTC), derived from the total phenolic compounds of Chinese dragon's blood, is now used in the treatment of ischemic stroke in convalescence. The aim of this study is to explore the neuroprotective effect of LTC from the perspective of neuroinflammation. Cell viability and lactate dehydrogenase (LDH) release were measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and LDH assay kit. Proinflammatory mediators and cytokines production including Nitric Oxide (NO), prostaglandin E2, (PGE2), interleukin (IL-β), IL-6, and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA) assay. In addition, western blot was used to detect the expression of inflammatory proteins associated with the mitogen-activated protein kinases (MAPKs), janus kinase/signal transducer and activator of tranions (JAK/STAT), nuclear transcription factor κB (NF-κB), and nuclear factor erythroid-2-related actor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathways. Moreover, immunofluorescence assay and electrophoretic mobility shift assays (EMSA) were performed to determine the Nrf2 translocation and the binding-DNA activity of NF-κB, respectively. LTC at 0.5 to 2 μg/mL significantly increased cell viability and decreased LDH, NO, PGE2, IL-1β, IL-6, and TNF-α production in oxygen-glucose deprivation/reoxygenation (OGD/R) and lipopolysaccharide (LPS)-induced BV2 microglia cells. Meanwhile, LTC not only decreased the protein expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) but also down-regulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38, and up-regulated HO-1 expression via nuclear translocation of Nrf2. LTC can significantly inhibit the phosphorylation of JAK1/STAT3 and reduce the translocation of NF-κB from cytosol to nucleus as well as the binding-DNA activity. PC12 cell pretreated with LTC-condition medium (CM) significantly alleviated LPS-induced neurotoxicity and increased PC12 cell viability in a dose-dependent manner. The present study showed that LTC exhibited a strong antineuroinflammatory activity and neuroprotective effects on LPS-stimulated BV2 microglial cells and PC12 cells.

Systems Pharmacology-Dissection of the Molecular Mechanisms of Dragon's Blood in Improving Ischemic Stroke Prognosis.

Materials and Methods (1) Based on system-pharmacology platform, the potential active compounds of DB are screened out according to ADME. (2) The ischemic stroke-related targets are predicted by utilizing these active compounds as probes, mapping the targets to the CTD database to establish a molecular-target-disease network. (3) To analyze the mechanism of DB treatment for the prognosis of ischemic stroke, we used the Metascape and DAVID databases to construct “ischemic stroke pathways”. (4) PC12 cells were used to explore the protective effect of loureirin B on oxygen-glucose deprivation/reperfusion (OGD/R) injury, and BV-2 cells were used to determine the anti-inflammation effect of 4′,7-dihydroxyflavone. Results Finally, we obtained 38 active compounds and 58 stroke-related targets. Network and pathway analysis indicate that DB is effective in the treatment of ischemic stroke by enhancing cell survival and inhibiting inflammatory and antiplatelet activation. In in vitro experiments, the main component loureirin B promoted the expression of HO-1 and Bcl-2 via positive regulation of PI3K/AKT/CREB and Nrf2 signaling pathways in PC12 cells against OGD/R damage. And the anti-inflammatory activity of 4′,7-dihydroxyflavone was related to the inhibition of COX-2, TNF-α, and IL-6 in LPS-induced BV-2 cells. Conclusions In our study, the results illustrated that DB in improving ischemic stroke prognosis may involve enhancing cell survival and antioxidant, anti-inflammation, and antiplatelet activities.

Effect of Longxuetongluo capsule in protecting vascular endothelial cells against oxygen-glucose deprivation/reoxygenation-induced apoptosis

Focal cerebral ischemia reperfusion is an essential process during ischemic stroke. The apoptosis of vascular endothelial cells induced by ischemia/reperfusion (I/R) injury is an important cause for brain injury after focal cerebral ischemia. Longxuetongluo capsule (LTC) has been used for the treatment of ischemic stroke in clinic. However, its underlying action mechanism is still unclear. This study aimed to verify the protective effect and mechanisms of LTC on HUVEC cells against oxygen-glucose deprivation/reoxygenation (OGD/R) injury through MTT, LDH, flow cytometry, AO/EB staining and western blot assays. As a result, OGD/R significantly decreased the viability of HUVEC cells, which was significantly improved by LTC. LDH release assay showed that OGD/R significantly increased the lactate dehydrogenase (LDH) release, and LTC dramatically reduced the OGD/R-induced LDH release. Further mechanism study indicated that LTC dose-dependently inhibited the cleavage of PARP, caspase 3, and caspase 9 induced by OGD/R, suggesting that LTC could inhibit the activation of caspase 3/9 apoptosis pathway in the OGD/R-induced apoptosis of HUVEC cells. In conclusion, LTC could protect HUVEC cells against OGD/R injury by inhibiting the activation of mitochondria-related caspase 3/9 apoptosis pathway.

Method development and application for multi-component quantification in rats after oral administration of Longxuetongluo Capsule by UHPLC–MS/MS

Although wide applications towards ischemic stroke in clinic, the therapeutic materials of Longxuetongluo Capsule (LTC) that is composed of total phenolic extract of Chinese dragon’s blood, are still largely unclear. Exposure pattern characterization of those drug-derived components in vivo, notably in circulation system has been recommended as a viable approach to disclose the effective components of a given herbal medicine. Herein, we aimed to develop a robust method being capable of multi-component quantification in either rat plasma or tissues following oral administration of LTC, and to clarify the kinetic profiles of 11 primary drug-derived phenolic derivatives. Proteins precipitation was carried out for the plasma as well as homogenized tissue samples with acetonitrile. Chromatographic separations were achieved using UHPLC equipped with a shim-pack XR-ODS II column, and confidence-enhanced detection was accomplished through the joint employment of selected-reaction monitoring and tandem mass spectrometry (SRM-MS/MS) on a hybrid triple quadrupole-linear ion trap mass spectrometer. Diverse validation assays proved the method to be sensitive, precise, and rapid for simultaneous determination of those 11 components. Pharmacokinetic and tissue distribution investigations were subsequently conducted in rat after a single 500 mg/kg oral dose. Rapid absorption (Tmax, 11.53–68.27 min) and elimination (T1/2, 6.893–57.90 min) occurred for all analytes-of-interest. Extensive occurrences were observed for 7,4′-dihydroxy-5-methoxyhomoisoflavanone (Cmax, 340.0 ng/mL), thevetiaflavone (Cmax, 42.86 ng/mL), 5,7,4′-trihydroxyhomoisoflavanone (Cmax, 41.55 ng/mL), and pterostilbene (Cmax, 25.49 ng/mL) in plasma. Significant distributions occurred for all analytes in the liver as well as kidney, and several compounds could be found in brain. The findings described are envisioned to provide promising information for the in-depth clarification of the therapeutic entities, and also to offer a practical approach for therapeutic drug monitoring of LTC in clinic.

Protective effect of Longxue Tongluo capsule on oxidized low-density lipoprotein damage to human umbilical vein endothelial cells

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 (P<0.01) increased, NO content and SOD activity significantly decreased (P<0.01, P<0.05), and the expressions of ICAM-1, VCAM-1, p-p65/p65 and p-IKB(P<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.

Anti-atherosclerotic effect of Longxuetongluo Capsule in high cholesterol diet induced atherosclerosis model rats

Chinese dragon’s blood, the red resin of Dracaena cochinchinensis, one of the famous traditional medicines, has been used to promote blood circulation, disperse blood stasis, stop bleeding, relieve pain and muscle regeneration for thousands of years. The aims of this study were to evaluate the anti-atherosclerotic effect of Longxuetongluo Capsule (LTC), which made by total phenolic compounds of Chinese dragon’s blood, in high cholesterol diet (HCD)-induced atherosclerosis model rats and explore the possible mechanism. Atherosclerosis rats were induced by administration of HCD for 4 weeks and treated with atorvastatin (2.08mg/kg/d) or various concentrations of LTC (81, 162 and 324mg/kg/d) for additional 4 weeks. Body weight (BW), lipid profiles, serum VCAM-1, ICAM-1, MCP-1, AST and ALT were then tested. Histopathological evaluation of aorta and liver were determined by hematoxylin and eosin staining. NF-κB expression in aorta was detected by Immunohistochemical staining. Meanwhile, the inhibition effects of LTC on the migration and proliferation and Intracellular Ca2+ levels induced by PDGF-BB were also evaluated in rat aortic smooth muscle cells (A7r5). The results demonstrated that LTC produced a significant anti-atherosclerotic activity in terms of reduction in serum lipids and lipoprotein profile, VCAM-1, ICAM-1, MCP-1, AST, ALT levels, and increase in HDL-c level compared to atherosclerotic group. Rats treated with LTC not only attenuated the pathological region and atheroma formation, but also reduced hepatic steatosis and inflammatory cell infiltration. Immunohistochemical analysis showed LTC reduced NF-κB expression in aorta. Furthermore, PDGF-BB induced proliferation and migration of A7r5 and intracellular calcium rise were also abrogated by LTC. The results indicate that LTC prevents atherosclerosis and fatty liver by controlling lipid metabolism, the underlying mechanism may attributed to its anti-inflammation activity, regulation of the vascular smooth muscle function and intracellular calcium signaling.

Characterization and quantitative analysis of phenolic derivatives in Longxuetongluo Capsule by HPLC-DAD-IT-TOF-MS

Longxuetongluo Capsule (LTC), which is derived from the total phenolic extract of Chinese dragon’s blood, has been proved to be safe as well as effective towards ischemic stroke. However, the effective material basis remains unclear. The present study thereby focused on the clarification of the qualitative and quantitative properties for the phenolic derivatives in LTC. Regarding homolog-focused chemical profiling, the mass fragmentation patterns of the primary subtypes of phenolic compounds such as homoisoflavanones, flavanes, chalcones, and flavonoid oligomers were summarized by assaying authentic references with hybrid ion trap time-of-flight mass spectrometry, and the chemical structures of 124 phenolic compounds, in total, were unambiguously or tentatively annotated in LTC by matching the accurate mass spectral profiles with the proposed mass cracking rules and those reference substances. Afterwards, simultaneous determination of 12 primary phenolic compounds was carried out in different batches of LTC using HPLC-DAD, after that the method was proved to be accurate, precise, and reproducible according to diverse method validation assays. The obtained findings are expected to be meaningful for clarifying the effective substances and quality assessment of LTC.

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

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.

Longxuetongluo capsule inhibits atherosclerosis progression in high-fat diet-induced ApoE−/− mice by improving endothelial dysfunction

Background and aimsChinese dragon's blood has been used to treat blood stasis for thousands of years. Its total phenolic extract (Longxuetongluo capsule, LTC) is used for the treatment of ischemic stroke; however, its protective effect against atherosclerosis remains poorly understood. This paper aims to investigate the antiatherosclerotic effect of LTC and the underlying mechanisms in high-fat diet (HFD)-induced ApoE−/− mice. MethodsThe levels of plasma lipid and areas of atherosclerotic lesions in the aortic sinus in ApoE−/− mice were evaluated. The effect of LTC on the nitric oxide (NO) production in oxidized low-density lipoprotein (ox-LDL)-stimulated human umbilical vein endothelial cells (HUVECs) was determined. The adhesion of monocytes to ox-LDL-stimulated HUVECs was further studied. ResultsLTC at low, medium, and high doses markedly decreased the atherosclerotic lesion areas of the aortic sinus in HFD-induced ApoE−/− mice by 26.4% (p < 0.05), 30.1% (p < 0.05), and 46.5% (p < 0.01), respectively, although it did not improve the dyslipidemia. Furthermore, LTC restored the diminished NO production of ox-LDL-stimulated HUVECs (p < 0.001) and inhibited the adhesion between monocytes and endothelial cells (p < 0.01). LTC appeared to alleviate ox-LDL-stimulated dysfunction of HUVECs, and inhibit the adhesion of monocytes to HUVECs via the MAPK/IKK/IκB/NF-κB signaling pathway, thus decrease atherosclerotic lesions in the aortic sinus in HFD-induced ApoE−/− mice. ConclusionsThese findings suggest the potential of LTC for use as an effective agent against atherosclerosis.

Longxuetongluo Capsule Improves Erythrocyte Function against Lipid Peroxidation and Abnormal Hemorheological Parameters in High Fat Diet-Induced ApoE-/- Mice.

Chinese dragon's blood, the red resin of Dracaena cochinchinensis, one of the renowned traditional medicines, has been used to facilitate blood circulation and disperse blood stasis for thousands of years. Phenolic compounds are considered to be responsible for its main biological activities. In this study, total phenolic compounds of Chinese dragon's blood were made into capsule (Longxuetongluo Capsule, LTC) and their effects on the abnormal hemorheological properties were examined by high fat diet (HFD) induced ApoE−/− mice. Compared to the model group, LTC recovered the abnormal hemorheological parameters in HFD-induced ApoE−/− mice by reducing whole blood viscosity (WBV) at high rate and improving erythrocyte function. In conclusion, LTC could ameliorate erythrocyte deformability and osmotic fragility through the reduction of lipid peroxidation on plasma and erythrocyte membranes in HFD-induced ApoE−/− mice, which supported the traditional uses of Chinese dragon's blood as an effective agent for improving blood microcirculation in hypercholesterolemia.