Tong Luo Jiu Nao Research Articles

Ability of Tong Luo Jiu Nao Oral Solution to Improve Cognitive and Gait Deficits in a Rat Model of Focal Cerebral Ischemia

ABSTRACT Objective To investigate the ability of Tong Luo Jiu Nao (TLJN) Oral Solution to improve cognitive and gait deficits in a rat model of focal cerebral ischemia. Methods An intraluminal suture method was used to create a rat model of focal cerebral ischemia. Experimental animals were divided into 4 groups: sham surgery, cerebral ischemia model, cerebral ischemia model+low-dose TLJN Oral Solution (150 mg/kg), cerebral ischemia model + high-dose TLJN Oral Solution (300 mg/kg). After 4 weeks of drug intervention, water maze and gait analyses were conducted. Brain tissue was stained with hematoxylin and eosin and used to monitor and calculate the volume ratio of cerebral infarction in each group of permanent middle cerebral artery occlusion (pMCAO) rats. Results (1) Gait analyses: TLJN Oral Solution significantly improved the overall gait of the focal cerebral ischemic rat model. The improvement in walking speed might have been achieved by the shortening of the animals’ stride time. The shortening of stance time by TLJN in the cerebral ischemic animals might be due to the shortening of brake and propulsion times, while the shortening of stride time by TLJN in the cerebral ischemic animals might have been due to the shortening of stance time and swing time. (2) Water maze navigation test: high-dose TLJN Oral Solution significantly reduced the animals’ total swimming distance and duration and increased the proportion of swimming distance and time spent in the target quadrant, suggesting that TLJN significantly improved the cognitive deficit in the animal model. (3) High-dose TLJN significantly reduced the cerebral infarction volume ratio of the pMCAO rats. Conclusion This intraluminal suture method can reliably replicate a rat model of focal cerebral ischemia. TLJN Oral Solution improves the gait and cognitive function of the focal cerebral ischemia rat model to various extents.

Tong Luo Jiu Nao, a Chinese Medicine Formula, Reduces Inflammatory Stress in a Mouse Model of Alzheimer's Disease.

The aim of this study is to extend the molecular mechanism of Tong Luo Jiu Nao (TLJN) for Alzheimer's disease (AD), which is a modern Chinese formula that has been used to treat AD. The senescence-accelerated mouse prone 8 strain (SAMP8) is one of the most appropriate models to study the mechanism that underlies AD. The levels of plasma amyloid β (Aβ) and the Aβ deposits were measured using enzyme-linked immunosorbent assay and immunohistochemistry. Immunoblotting was used to observe the effect of TLJN on inflammatory mediator expression in an senescence-accelerated mouse model of AD. Our data showed that the TLJN-treated groups exhibited a reduction in plasma Aβ levels and reduced Aβ expression. Moreover, TLJN effectively attenuated Aβ-induced activation of extracellular signal-regulated kinase and c-Jun N-terminal kinases and blocked changes in inflammatory mediator expression. These data suggest that TLJN might have protective effects and could potentially act to attenuate inflammatory stress in the pathogenesis of AD.

Tong Luo Jiu Nao ameliorates Aβ1-40-induced cognitive impairment on adaptive behavior learning by modulating ERK/CaMKII/CREB signaling in the hippocampus.

BackgroundTong Luo Jiu Nao (TLJN), a modern formula of Chinese medicine extracts on the basis of Traditional Chinese Medicine theory, has been used to treat dementia. The present study aimed to investigate its ameliorating effects on Aβ1–40-induced cognitive impairment in rats using a series of novel reward-directed instrumental learning (RDIL) tasks, and to determine its possible mechanism of action.MethodsRats were pretreated with TLJN extract (0.9 and 1.8 g/kg, p.o.) for 10 daysbefore surgery, and were trained to gain reward reinforcement by lever pressing at the meantime. Thereafter, rats received a bilateral microinjection of Aβ1–40 in CA1 regions of the hippocampus. Cognitive performance was evaluated with the goal directed (higher response ratio) and habit (visual signal discrimination and extinction) learning tasks, as well as on the levels of biochemical parameters and molecules.ResultsOur findings first demonstrated that TLJN can improve Aβ1–40-induced amnesia in RDIL via enhancing the comprehension of action-outcome association and the utilization of cue information to guide behavior. Then, its ameliorating effects should attribute to the modulation of ERK/CaMKII/CREB signaling in the hippocampus.ConclusionTLJN can markedly enhance cognitions of Aβ1–40 microinjection animal model in adaptive behavioral tasks. It has the potential, possibly as complementary and alternative therapy, to prevent and/or delay the deterioration of cognitive impairment in AD.

Chinese herbal formula Tongluo Jiunao injection protects against cerebral ischemia by activating neurotrophin 3/tropomyosin-related kinase C pathway.

The Chinese herbal formula Tongluo Jiunao, containing the active components Panax notoginseng and Gardenia jasminoides, has recently been patented and is in use clinically. It is known to be neuroprotective in cerebral ischemia, but the underlying pathway remains poorly understood. In the present study, we established a rat model of cerebral ischemia by occlusion of the middle cerebral artery, and administered Tongluo Jiunao, a positive control (Xuesai Tong, containing Panax notoginseng) or saline intraperitoneally to investigate the pathway involved in the action of Tongluo Jiunao injection. 2,3,5-Triphenyltetrazolium chloride (TTC) staining showed that the cerebral infarct area was significantly smaller in model rats that received Tongluo Jiunao than in those that received saline. Enzyme-linked immunosorbent assay revealed significantly greater expression of neurotrophin 3 and growth-associated protein 43 in ischemic cerebral tissue, and serum levels of neurotrophin 3, in the Tongluo Jiunao group than in the saline group. The reverse transcription polymerase chain reaction and immunohistochemical staining showed that after treatment with Tongluo Jiunao or Xuesai Tong, tropomyosin-related kinase C gene expression and immunoreactivity were significantly elevated compared with saline, with the greatest expression observed after Tongluo Jiunao treatment. These findings suggest that Tongluo Jiunao injection exerts a neuroprotective effect in rats with cerebral ischemia by activating the neurotrophin 3/tropomyosin-related kinase C pathway.

Antianxiety effect of Tong Luo Jiu Nao, traditional Chinese medicinal preparation, in rats

Tong Luo Jiu Nao (TLJN), a modern formula of traditional Chinese medicine, has proven to be clinically efficacious in several vascular cerebral diseases but its specific effect is not known. In the present study we investigated the acute and persisting effects of TLJN on anxiety model in Wistar male rats. TLJN was administered intragastrically during three successive days (Days 1–3) and then, the double TLJN dose was given on Day 7. For the evaluation of anxiety-related behavior of animals, we used the open field (OF) and elevated plus maze (EPM) paradigms. Testing in the OF was performed on Days 1, 2, 3, 4, 8, 14 and 22; in the EPM on the Day 23.Two-way repeated-measures ANOVA revealed significant differences between control and TLJN treated animals in the open field model where TLJN induced increased exploratory activity, indicating reduced anxiety-like behavior. The effect persisted for several days after the treatment and was still present on Days 14 and 22. Reduced anxiety-like behavior was also observed on EPM 16 days after the last TLJN administration.The results demonstrate behavioral effects of intragastric administered TLJN, which indicate reduced anxiety. Persistence of the induced behavioral changes suggests prolonged duration of action.

The herbal compound geniposide rescues formaldehyde-induced apoptosis in N2a neuroblastoma cells

The herbal medicine Tong Luo Jiu Nao (TLJN) contains geniposide (GP) and ginsenoside Rg1 at a molar ratio of 10:1. Rg1 is the major component of another herbal medicine, panax notoginseng saponin (PNS). TLJN has been shown to strengthen brain function in humans, and in animals it improves learning and memory. We have previously shown that TLJN reduces amyloidogenic processing in Alzheimer's disease (AD) mouse models. Together this suggests TLJN may be a potential treatment for patients with dementia. Because chronic damage of the central nervous system by formaldehyde (FA) has been presented as a risk factor for age-associated cognitive dysfunction, in the present study we investigated the protective effect of both TLJN and GP in neuron-like cells exposed to FA. FA-exposed murine N2a neuroblastoma cells were incubated with TLJN, its main ingredient GP, as well as PNS, to measure cell viability and morphology, the rate of apoptosis and expression of genes encoding Akt, FOXO3, Bcl2 and p53. The CCK-8 assay, cytoskeletal staining and flow cytometry were used to test cell viability, morphology and apoptosis, respectively. Fluorescent quantitative real-time PCR (qRT-PCR) was used to monitor changes in gene expression, and HPLC to determine the rate of FA clearance. Treatment of N2a cells with 0.09 mmol L(-1) FA for 24 h significantly reduced cell viability, changed cell morphology and promoted apoptosis. Both TLJN and GP conferred neuroprotection to FA-treated N2a cells, whereas PNS, which had to be used at lower concentrations because of its toxicity, did not. Our data demonstrate that TLJN can rescue neuronal damage caused by FA and that its main ingredient, GP, has a major role in this efficacy. This presents purified GP as a drug or lead compound for the treatment of AD.

Geniposide, the component of the Chinese herbal formula Tongluojiunao, protects amyloid-β peptide (1-42-mediated death of hippocampal neurons via the non-classical estrogen signaling pathway.

Tongluojiunao (TLJN) is an herbal medicine consisting of two main components, geniposide and ginsenoside Rg1. TLJN has been shown to protect primary cultured hippocampal neurons. However, its mechanism of action remains unclear. In the present study, primary cultured hippocampal neurons treated with Aβ1–42 (10 µmol/L) significantly increased the release of lactate dehydrogenase, which was markedly reduced by TLJN (2 µL/mL), specifically by the component geniposide (26 µmol/L), but not ginsenoside Rg1 (2.5 µmol/L). The estrogen receptor inhibitor, ICI182780 (1 µmol/L), did not block TLJN- or geniposide-mediated decrease of lactate dehydrogenase under Aβ1–42-exposed conditions. However, the phosphatidyl inositol 3-kinase or mitogen-activated protein kinase pathway inhibitor, LY294002 (50 µmol/L) or U0126 (10 µmol/L), respectively blocked the decrease of lactate dehydrogenase mediated by TLJN or geniposide. Therefore, these results suggest that the non-classical estrogen pathway (i.e., phosphatidyl inositol 3-kinase or mitogen-activated protein kinase) is involved in the neuroprotective effect of TLJN, specifically its component, geniposide, against Aβ1–42-mediated cell death in primary cultured hippocampal neurons.

Neuroprotective effects of geniposide in SH-SY5Y cells and primary hippocampal neurons exposed to Aβ42.

Our former studies have suggested that TongLuoJiuNao (TLJN) is clinically efficacious in the treatment of dementia and improving learning and memory in AD models. When Aβ aggregated with oligomer, it is known to be able to induce cellular toxicity as well as cognitive impairment. We tested the possibility that TLJN affects the formation of Aβ oligomers. In our experiment, TLJN improved cell viability, inhibited LDH release, and promoted the outgrowth of neurites of neurons treated with Aβ. Geniposide, the main component of TLJN, could increase the cell viability of SY5Y-APP695sw cells. The cytotoxicity of pretreated Aβ with geniposide was decreased in a dose-dependent manner. SDS-PAGE and Western blotting showed that geniposide and TLJN stimulated Aβ oligomer assembly. Compared with the control, more and longer fibrils of Aβ in the presence of geniposide were observed under electron microscope though the fibrils became less sensitive to thioflavin T staining. In sum, geniposide is able to protect neurons from Aβ-induced damage by remodeling Aβ.

The protective effect of geniposide on human neuroblastoma cells in the presence of formaldehyde

BackgroundFormaldehyde can induce misfolding and aggregation of Tau protein and β amyloid protein, which are characteristic pathological features of Alzheimer’s disease (AD). An increase in endogenous formaldehyde concentration in the brain is closely related to dementia in aging people. Therefore, the discovery of effective drugs to counteract the adverse impact of formaldehyde on neuronal cells is beneficial for the development of appropriate treatments for age-associated cognitive decline.MethodsIn this study, we assessed the neuroprotective properties of TongLuoJiuNao (TLJN), a traditional Chinese medicine preparation, against formaldehyde stress in human neuroblastoma cells (SH-SY5Y cell line). The effect of TLJN and its main ingredients (geniposide and ginsenoside Rg1) on cell viability, apoptosis, intracellular antioxidant activity and the expression of apoptotic-related genes in the presence of formaldehyde were monitored.ResultsCell counting studies showed that in the presence of TLJN, the viability of formaldehyde-treated SH-SY5Y cells significantly recovered. Laser scanning confocal microscopy revealed that the morphology of formaldehyde-injured cells was rescued by TLJN and geniposide, an effective ingredient of TLJN. Moreover, the inhibitory effect of geniposide on formaldehyde-induced apoptosis was dose-dependent. The activity of intracellular antioxidants (superoxide dismutase and glutathione peroxidase) increased, as did mRNA and protein levels of the antiapoptotic gene Bcl-2 after the addition of geniposide. In contrast, the expression of the apoptotic-related gene - P53, apoptotic executer - caspase 3 and apoptotic initiator - caspase 9 were downregulated after geniposide treatment.ConclusionsOur results indicate that geniposide can protect SH-SY5Y cells against formaldehyde stress through modulating the expression of Bcl-2, P53, caspase 3 and caspase 9, and by increasing the activity of intracellular superoxide dismutase and glutathione peroxidase.

Chronic Administration of Anti-Stroke Herbal Medicine TongLuoJiuNao Reduces Amyloidogenic Processing of Amyloid Precursor Protein in a Mouse Model of Alzheimer’s Disease

Composed of Ginsenoside Rg1 and Geniposide, the herbal medicine TongLuoJiuNao (TLJN) injection liquid has anti-inflammatory properties and can improve learning and memory in mice. Recently, TLJN has been used to treat the patients with cerebral ischemic stroke and vascular dementia, which significantly increase the risk of developing Alzheimer’s disease (AD) in the early human beings. Although beneficial effects of TLJN have been reported in the vascular-associated brain disorders, the roles of TLJN in AD brains are still not clear. In this study, we chronically administered TLJN in amyloid precursor protein (APP) Swedish mutant transgenic mice (APP23) from 6 months old of age, which is at the onset of Aβ plaques, to 12 months old. We found that TLJN significantly decreased Aβ production and deposition in the brain of APP23 mice. Furthermore, we observed that TLJN down-regulated the levels and activity of β-secretase 1 (BACE1) protein as well as the expression levels of γ-secretase complex components: PS1, nicastrin and anterior pharynx-defective 1 (APH1) but not presenilin enhancer 2 (PEN2). The results suggest an inhibitory effect of TLJN on amyloidogenic APP processing by down-regulating the cleavage enzymes BACE1 and γ-secretase.

Tongluo Jiunao Injection, a Chinese herbal fomula protects the focal ischemic brain injury and decreases the expression of Semaphorin 3A and its receptor Neuropilin 1

Tongluo Jiunao (TLJN) injection has been shown to exhibit protective effects following ischemia injury in vivo and in vitro. We hypothesized that TLJN injection alleviates the injury of neuron and brain blood barrier by inhibiting the expression of Semaphorin 3A (Sema 3A) and its receptor Neuropilin 1 (Nrp‐1) following ischemia stroke. A permanent middle cerebral artery occlusion (PMCAO) surgery was induced in the study on SD rats. Brain edema, infarct volume and spatialtemporal expression of Sema 3A and Nrp‐1 were detected. Triphenyltetrazolium chloride staining results revealed that TLJN injection reduced the infarct volume from 1d to 7d (p<0.01) and edema of brain at 1d (p<0.01) post injury compared to model group. Double‐labeled immunofluorescence showed that Sema 3A and Nrp‐1 positive cells increased in cortex and hippocampus in ipsilateral hemisphere at all the time points after the onset of PMCAO. Enzyme‐linked immunosorbent assay results showed that TLJN decreased Sema 3A. Reverse‐transcription polymerase chain reaction and western blot results showed that TLJN injection decreased the expression of Sema 3A and Nrp‐1 in the ischemic brain tissues of rats. These results suggested that TLJN injection reduced the damage of brain by decreasing the infarction volumn and edema of brain which is related to its inhibitory of Sema 3A and Nrp‐1.

Effect of Tong Luo Jiu Nao on Aβ-degrading enzymes in AD rat brains

Ethnopharmacological relevance Tong Luo Jiu Nao (TLJN) is a modern Chinese formula based on Traditional Chinese Medicine theory that has been used to treat ischemic cerebral stroke and vascular dementia. TLJN belongs to the ethnopharmacological family of medicines. In this study, we investigated the mechanism of the TLJN effect on Alzheimer's disease (AD). Aim of the study To investigate the effect of TLJN on β-amyloid-degrading enzymes and learning and memory in the AD rat brain. Materials and methods AD rats whose disease was induced by Aβ 25–35 injection into the bilateral hippocampus CA1 region were subjected to intragastric administration of various preparations. The experimental animals were healthy male Sprague-Dawley rats which were randomly divided into normal, sham, model, TLJN min, TLJN max and donepezil hydrochloride groups. Spontaneous alternation and passive avoidance behavior, which are regarded as measures of spatial learning and memory, were investigated using Y-maze testing. Western blotting and immunohistochemistry were used to observe the therapeutic effect of TLJN on the deposits of amyloid plaque and on the expression of synaptophysin, insulin-degrading enzyme and neprilysin. Results Y-maze results showed that the AD model group presented with spatial learning and memory impairments. Hematoxylin–eosin and Congo red staining indicated neuronal impairment and deposits of amyloid plaque in the model group and these results were consistent with their learning and memory deficits in the Y-maze. The TLJN-treated groups exhibited prolonged a cavity delitescence, decreased arm entries and improvement in learning and memory. Moreover, the structure of the neurons of the treated groups was restored and the expression of synaptophysin increased in both the hippocampus and cortex. In addition, their levels of insulin-degrading enzyme and neprilysin in the cortex and hippocampus were upregulated and the amyloid plaque was decreased. Conclusion TLJN can improve learning and memory, up-regulate insulin-degrading enzyme and neprilysin levels, promote the degrading of Aβ and clear amyloid plaque from the AD rat brain. In future, TLJN may have significant therapeutic potential in the treatment of AD patients.

Effects of Tongluo-Jiunao Injection on Cerebral Ischemia–Reperfusion Injury Rats

Abstract The aim of this study was to investigate the protective effects of Tongluo-Jiunao (TLJN) injection on brain tissues of focal cerebral ischemia-reperfusion injury rats. Cerebral ischemia-reperfusion injury rat model was established by middle cerebral artery occlusion. All animal models were divided into the following four groups: sham, model, TLJN, and edaravone groups. The brain infarct volume, concentration of IGF-1 in brain tissues and serum were detected at 24 h, 3 d, and 7 d after cerebral ischemia-reperfusion injury. The results showed that compared to the model group, the brain infarct volume of TLJN group was significantly reduced, and the expression of IGF-1 of cerebral cortex was upregulated in TLJN group. It was concluded that TLJN has significant protective effects on brain tissues in focal cerebral ischemia-reperfusion injury rat, and the IGF-1 may play an important role in the protective effects of brain tissues.

Brain microvascular endothelial cells mediate neuroprotective effects on ischemia/reperfusion neurons

Aim of the study The permeability of the blood–brain barrier (BBB) is a bottleneck for the development of new cerebropathy medications because the medication must be transmitted across the BBB to achieve its curative function. To explore a new approach to the treatment of brain disease, this study investigated the mediating effects of brain microvascular endothelial cells (MVECs) on injured neurons. Materials and methods MVECs and cortical neurons were cultured and damage by cerebral ischemia/reperfusion (I/R) was simulated. The conditioned media from four groups of MVECs – normal cells (N-CM), normal cells treated with Tong Luo Jiu Nao (TLJN) (NT-CM), simulated cerebral I/R cells (I/R-CM), and simulated cells treated with TLJN (I/RTCM) – were then collected. These conditioned media were added to neuronal cultures and the viability of the neurons was examined. Results The results demonstrated that N-CM could alleviate I/R damage to neurons, and this capacity could be improved by TLJN treatment. However, I/R-CM could cause damage to normal and I/R neurons, while I/RT-CM could significantly alleviate the damage to I/R neurons. Conclusions We propose that MVECs secrete active substances that influence the survival of neurons, and so MVECs may mediate a neuroprotective effect on ischemia/reperfusion neurons.

Conditioned mediums of different rat cerebral microvascular endothelial cells against damage of ischemia and ischemia/reperfusion neurons

Using the method of lactate dehydrogenase (LDH) assay, to observe the activities of rat cerebral microvascular endothelial cells (CMECs) intervened by Tongluo Jiunao Injection (TLJNI), a traditional Chinese compound drug removing toxin to dredge brain collaterals, and then further study the effects of different kinds of conditioned mediums (CMECs-CM) of cerebral microvascular endothelial cells on ischemia and ischemia/reperfusion cerebral cortex cells, and to probe into the drug pharmacological mechanisms of CMECs in modulating the neurons. Three kinds of CMECs (normal, ischemic and ischemic/reperfusional) were all treated by TLJNI previously, and then the three pairs of CMECs-CM without serum were collected respectively for LDH assay. Rat cerebral cortex neurons were also primarily cultured and then divided into similar three groups (normal, ischemic and ischemic/reperfusional). The neuron responses caused by CMECs-CM at different concentrations were observed by using LDH transudation rate assay. The LDH release values of ischemic and ischemic/reperfusional CMECs with TLJNI treatment were obviously reduced (P<0.01) compared with the same kinds of CMECs untreated. For ischemic neurons, both conditioned medium of ischemic CMECs (Is-CM) and conditioned medium of ischemic CMECs with drug treatment (IsT-CM) in high concentration of 100% increased the LDH transudation rate (P<0.01), while in low concentration of 10%, IsT-CM reduced the transudation rate (P<0.05). For ischemia/reperfusion neurons, all kinds of CMECs-CM reduced the transudation rate respectively (P<0.05 or P<0.01). As far as each group concentration was concerned, 10% or 50% showed relatively stronger effects, and both conditioned medium of normal CMECs (N-CM) group and conditioned medium of ischemic/reperfusional CMECs (Rp-CM) group had statistical significance (P<0.05 or P<0.01). For normal neurons, all kinds of CMECs-CM increased the transudation rate respectively (P<0.05 or P<0.01). As far as each group concentration was concerned, only conditioned medium of normal CMECs (N-CM) had statistical significance (P<0.05 or P<0.01). The study shows that TLJNI is capable of preventing the damage of CMECs from both ischemia and ischemia/reperfusion states. Chinese drug can restrain the brain ischemia and ischemia/reperfusion damage by the media that CMECs modulate the neurons, demonstrating the pharmacological mechanisms of TLJNI. This work also indicates that there exist some active substances against ischemia/reperfusion injury secreted from CMECs-CM with TLJNI treatment.