Huifen Zhou

PET Demonstrates Functional Recovery after Treatment by Danhong Injection in a Rat Model of Cerebral Ischemic-Reperfusion Injury

This study aimed to investigate neuroprotection of Danhong injection (DHI) in a rat model of cerebral ischemia using 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET). Method. Rats were divided into 5 groups: sham group, ischemia-reperfusion untreated (IRU) group, DHI-1 group (DHI 1 mL/kg/d), DHI-2 group (DHI 2 mL/kg/d), and DHI-4 group (DHI 4 mL/kg/d). AII the treated groups were intraperitoneally injected with DHI daily for 14 days. The therapeutic effects in terms of cerebral infarct volume, neurological function, and cerebral glucose metabolism were evaluated. Expression of TNF-α and IL-1β was detected with enzyme-linked immunosorbent assay (ELISA). Levels of mature neuronal marker (NeuN), glial marker (GFAP), vascular density factor (vWF), and glucose transporter 1 (GLUT1) were assessed by immunohistochemistry. Results. Compared with the IRU group, rats treated with DHI showed dose dependent reductions in cerebral infarct volume and levels of proinflammatory cytokines, improvement of neurological function, and recovery of cerebral glucose metabolism. Meanwhile, the significantly increased numbers of neurons, gliocytes, and vessels and the recovery of glucose utilization were found in the peri-infarct region after DHI treatment using immunohistochemical analysis. Conclusion. This study demonstrated the metabolic recovery after DHI treatment by micro-PET imaging with 18F-FDG and the neuroprotective effects of DHI in a rat model of cerebral ischemic-reperfusion injury.

Protective effect of Danhong Injection combined with Naoxintong Capsule on cerebral ischemia-reperfusion injury in rats

ETHNOPHARMACOLOGICAL RELEVANCE Danhong Injection (DHI) and Naoxintong Capsule (NXT) are renowned traditional Chinese medicine in China. The drug combination of DHI and NXT is frequently applied for the treatment of cardiovascular and cerebrovascular diseases in clinic. However, there had been no pharmacological experiment studies of interaction between DHI and NXT. Due to the drug interactions, exploring their interaction profile is of great importance. MATERIAL AND METHODS In this study, focal cerebral I/R injury in adult male Sprague-Dawley rats were induced by transient middle cerebral artery occlusion (tMCAO) for 1h followed by reperfusion. Rats were divided into 5 groups: sham group, ischemia reperfusion untreated group (IRU), DHI group (DHI 10mL/kg/d), NXT group (NXT 0.5g/kg/d), DHI plus NXT group (DHI-NXT, DHI 10mL/kg/d plus NXT 0.5g/kg/d). All drug-treated groups were respectively successive administrated for 7 days after ischemia/ reperfusion (I/R) injury. The effects on rat neurological function were estimated by neurological defect scores. Brain infarct volumes were determined based on 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Pathological changes in brain tissues were observed using hematoxylin and eosin (H&E) staining and transmission electron microscope (TEM). Levels of nitric oxide (NO), granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) in serum were determined with enzyme-linked immunosorbent assay (ELISA). Immunohisto-chemistry and Western blot were used to detect the expressions of basic fibroblast growth factor (bFGF), von Willebrand factor-microvessel vascular density (vWF-MVD), vascular endothelial cell growth factor (VEGF), transforming growth factor-β1 (TGF-β1), angiogenin-1 (Ang-1), angiogenin-2 (Ang-2) and platelet derived growth factor (PDGF) at day 7 after ischemia/reperfusion (I/R) injury. RESULTS Compared with IRU group and mono-therapy group (DHI group or NXT group), Danhong Injection combined with Naoxintong Capsule (DHI-NXT) group significantly ameliorated neurological deficits scores, infarct volume and pathological change, significantly decreased the overexpression of NO and the level of Ang-1, significantly increased the expressions of VEGF, Ang-2, G-CSF, GM-CSF, bFGF, PDGF, vWF, TGF-β1. CONCLUSION The protective benefits on rat brain against I/R injury were clearly produced when DHI and NXT were used in combination, which provided rational guidance for clinical combined application of DHI and NXT, and this protection maybe associated with the up-regulation expressions of the related chemokines and growth factors of angiogenesis.

Effects of Active Components of Fuzi and Gancao Compatibility on Bax, Bcl-2, and Caspase-3 in Chronic Heart Failure Rats

Hypaconitine (HA) and glycyrrhetinic acid (GA) are active components of Fuzi (Aconitum carmichaelii) and Gancao (Glycyrrhiza uralensis Fisch); they have been used in compatibility for chronic heart failure (CHF) from ancient times. The purpose of the present research was to explore whether apoptosis pathways were related with the protective effects of HA + GA against CHF rats or not. The rats were progressed with transverse-aortic constriction (TAC) operation for 4 weeks to build the CHF state, and then the Digoxin (1 mg/kg), HA (2.07 mg/kg), GA (25 mg/kg), and HA (2.07 mg/kg) + GA (25 mg/kg) were orally administrated to rats for 1 week. The levels of BNP and cTnI in the plasma were decreased in the HA + GA group, and the heart/body weight ratio (H/B) and left ventricular (LV) parameters of transthoracic echocardiography were also declined; moreover, the expressions of Bax, Bcl-2, and caspase-3 were all improved in the HA + GA group than other groups in the immunohistochemistry and western blot methods. In general, the data suggested that Fuzi and Gancao compatibility could protect the CHF rats from apoptosis, which provided a strong evidence for further searching for mechanisms of them.

Protective effect of hydroxysafflor yellow A alone or in combination with acetylglutamine on cerebral ischemia reperfusion injury in rat: A PET study using 18 F-fuorodeoxyglucose

Abstract Hydroxysafflor yellow A (HSYA) and acetylglutamine (NAG) are extensively applied in the treatment of brain injury. In this study, we investigated the neuroprotective effect and underlying mechanism of HSYA alone or together with NAG using a rat model of cerebral ischemia reperfusion injury. Male Sprague‐Dawley (SD) rats (n = 5) were intraperitoneally injected with 5, 10, 20 mg/kg HSYA, 300 mg/kg NAG and 10 mg/kg HSYA+300 mg/kg NAG after the onset of reperfusion and once each day for the following 7 days. After assessing the neurological deficit and infarct volume, we used 18F‐FDG‐PET to evaluate the regional cerebral metabolic rate of glucose consumption, immunohistochemical analysis to detect the expression of GFAP, NGF, Bcl‐2, Bax, caspase‐3 and ICAM‐1 in brain tissue at day 7 after cerebral I/R injury. Meanwhile, the mRNA levels of ICAM‐1, IL‐1ß, TNF‐&agr; and NF‐&kgr;B were determined by qRT‐PCR, the protein levels of Bcl‐2, Bax and caspase‐3 were detected by western blot. The results indicated that HSYA significantly up‐regulated glucose metabolism, improved neurological function, decreased cerebral infarction volume. HSYA alone or together with NAG attenuated apoptosis and inflammation by up‐regulating GFAP, NGF and Bcl‐2 expression, suppressing the expression of Bax, caspase‐3 and ICAM‐1, IL‐1ß, TNF‐&agr; and NF‐&kgr;B. These finding suggested that HSYA exerted neuroprotection against cerebral I/R injury by modulating inflammation and apoptosis process, and HSYA in combination with NAG possessed a synergetic effect on protecting cerebral I/R brain injury.

Glycyrrhetinic acid protects H9c2 cells from oxygen glucose deprivation-induced injury through the PI3K/AKt signaling pathway

Glycyrrhetinic acid (GA) is an ingredient of triterpene saponins found in Gancao (Radix Glycyrrhizae). Here, we investigated the protective effects of GA in H9c2 cells, and explored its possible mechanism of action. Different concentrations of GA were used to treat H9c2 cells under oxygen glucose deprivation. We analyzed cell necrosis and apoptosis using optical microscopy, Hoechst 33342 staining, FITC-annexin V/PI double-staining and lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB) and interleukin (IL)-1β assays. Changes in related pro-apoptosis and anti-apoptosis proteins were detected by Western blot. Optical microscopy showed that GA improved cell morphology, including cell shrinkage, cauliflower-like membrane blebbing, and even some cell debris. Meanwhile, GA also ameliorated cell nuclei characteristics such as nucleus size, chromatin condensation and bright staining from Hoechst 33342 staining. GA also lowered the apoptotic rate and the levels of LDH, CK-MB and IL-1β in a dose-dependent manner. Furthermore, GA treatment increased Bcl-2 protein expression and decreased caspase-8 and Bax protein expression, while elevating the Bcl-2/Bax ratio. GA preconditioning increased p-AKt protein expression; however, after adding LY 294002, the p-AKt expression decreased obviously. Our results demonstrated that GA could protect H9c2 cells from apoptosis in a dose-dependent manner, and the potential mechanism might be related to the PI3K/AKt signaling pathway.

Protective effects of Yinhuapinggan granule on mice with influenza viral pneumonia.

Yinhuapinggan granule (YHPG), a Chinese medicine granule on the basis of Ma-Huang-Tang (Ephedra Decoction) and the clinical experience of Professor Wan Haitong, has been shown to inhibit the growth of influenza virus in vitro. The aim of this study was to investigate the protective effects of YHPG on mice with influenza viral pneumonia and its effects on regulating related inflammatory cytokines in influenza virus A-infected mice. ICR mice were inoculated intranasally with 15 LD50 viral dose of influenza virus A/PR/8/34 (H1N1) and treatments with YHPG (7.5, 15 and 30g/kg) were orally administrated daily for 5 consecutive days after challenge, respectively. The results showed that mortality rate, lung index, lung histopathological changes, IL-6 and TNF-α in serum were significantly attenuated in the treatment of YHPG (15 and 30g/kg) than those in the IFV control group, while the levels of IL-2 was significantly enhanced. Moreover, the RT-PCR results revealed that YHPG (15 and 30g/kg) significantly depressed the expressions of IL-1β, IFN-γ and TNF-α mRNA in lung tissues. Furthermore, the immunohistochemical staining results also revealed that the expression of NF-κB p65 proteins was downregulated when treated with YHPG (15 and 30g/kg). These results showed YHPG has protective effects on IFV-infected mice, due to its ability of alleviation of lung damage, regulation of the cytokine production via inhibiting the NF-κB p65 activation, attenuation of systemic and pulmonary inflammatory responses.

Pharmacokinetics of Active Components From Guhong Injection in Normal and Pathological Rat Models of Cerebral Ischemia: A Comparative Study.

Background and Objectives: Guhong Injection (GHI) is usually administered for the treatment of stroke in clinics. Aceglutamide and hydroxyl safflower yellow A (HSYA) are its key ingredients for brain protective effect. To investigate the pharmacokinetics of aceglutamide and HSYA under pathological and normal conditions, the pharmacokinetic parameters and characteristics of middle cerebral artery occlusion (MCAO) and normal rats given the same dosage of GHI were studied compared. Methods: 12 SD rats were divided into two groups, namely, MCAO and normal groups. Both groups were treated with GHI in the same dosage. Plasma samples were collected from the jaw vein at different time points and subsequently tested by high-performance liquid chromatography (HPLC). Results: After administration of GHI, both aceglutamide and HSYA were immediately detected in the plasma. Ninety percent of aceglutamide and HSYA was eliminated within 3 h. For aceglutamide, statistically significant differences in the parameters including AUC(0−t), AUC(0−∞), AUMC(0−t), AUMC(0−∞), Cmax (P < 0.01), and Vz (P < 0.05). Meanwhile, compared with the MCAO group, in the normal group, the values of AUC(0−t), AUMC(0−t), VRT(0−t), and Cmax (P < 0.01) for HSYA were significantly higher, whereas the value of MRT(0−t) was significantly lower in the normal group. Conclusions: The in vivo trials based on the different models showed that, the pharmacokinetic behaviors and parameters of aceglutamide and HSYA in GHI were completely different. These results suggest that the pathological damage of ischemia-reperfusion has a significant impact on the pharmacokinetic traits of aceglutamide and HSYA.

Antiviral effects of Ma Huang Tang against H1N1 influenza virus infection in vitro and in an ICR pneumonia mouse model

Ma Huang Tang (MHT), a classical Chinese herbal decoction which has been used in clinic for thousands of years, was very effective in treating the upper respiratory tract infection. But its activity against influenza virus A, the anti-inflammatory effect and the underlying mechanisms have been poorly investigated in previous researches. In this study, the antiviral efficacy of MHT directly inhibiting influenza virus A was investigated in vitro in MDCK cells. In an ICR pneumonia mouse model infected with influenza virus A PR/8/34, MHT (8, 4 and 2 g/kg) were oral administrated for 7 days after viral challenge, to evaluate the effect of MHT on ameliorating viral pneumonia and decipher the underlying mechanisms. The in vitro results showed that MHT possessed antiviral activity with low toxicity. The in vivo assays showed that MHT (8 and 4 g/kg) significantly attenuated lung histopathological changes, decreased lung index, interleukin (IL)-4,5, tumor necrosis factor alpha (TNF-α), CD3+, CD8+ T cell levels, increased IL-2, gamma interferon (IFN-γ), CD4+ T cell levels and CD4+/CD8+ ratio, inhibited toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor associated factor 6 (TRAF6) protein levels. All these results demonstrate that MHT can strikingly ameliorate influenza virus A pneumonia in mice, which is associated with the regulating effect of MHT in the imbalance of bodys immune function and the MyD88-dependent signaling pathway of TLR4.

Protective mechanisms of hypaconitine and glycyrrhetinic acid compatibility in oxygen and glucose deprivation injury

This study investigated the protective effect of the compatibility of hypaconitine (HA) and glycyrrhetinic acid (GA) on H9c2 cells under oxygen and glucose deprivation (OGD)-induced injury, and the possible mechanisms. We found that HA+GA significantly improved pathology and morphology of the nucleus and ultrastructure of H9c2 cells under OGD as determined by Hoechst 33342 staining and transmission electron microscopy (TEM) tests. It also reduced the releases of lactate dehydrogenase (LDH), creatine kinase-myocardial band isoenzyme (CK-MB), and aspartate transaminase (AST) from the cultured supernatant of H9c2 cells, which were tested by enzyme-linked immune sorbent assay (ELISA) kits. In addition, it lessened the apoptotic rate as determined by a fluorescein isothiocyanate-annexin V/propidium iodide (FITC-AV/PI) double staining assay. It was also found that HA+GA might regulate the protein expression associated with the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Overall, the study demonstrated that HA+GA protected H9c2 cells against OGD-induced injury, and the signaling mechanism might be related to the PI3K/Akt signaling pathway.中文概要题目次乌头碱配伍甘草次酸对缺氧缺糖损伤H9c2 心肌细胞的保护作用及其分子机制目的探讨次乌头碱(HA)配伍甘草次酸(GA)对缺 氧缺糖(OGD)损伤H9c2 心肌细胞的保护作用 及其作用机制。创新点首次在OGD 模型中证明HA+GA 对H9c2 心肌细 胞有明显的保护作用。此作用与减少细胞坏死和 凋亡有关系,且其作用机制与磷脂酰肌醇-3-激酶/ 蛋白质丝氨酸-苏氨酸激酶(PI3K/Akt)信号通路 有关。方法采用H9c2 心肌细胞为研究对象,将其分为七组: 正常组、OGD 模型组、OGD+HA 组、OGD+GA 组、OGD+HA+GA 组、OGD+LY294002 组、 OGD+HA+GA+LY294002 组。采用Hoechst 33342 染色荧光显微镜及透射电镜观察前五组的H9c2 心肌细胞的形态学改变;采用酶联免疫吸附测定 法(ELISA)检测前五组细胞上清液中乳酸脱氢 酶(LDH)、肌酸激酶同工酶(CK-MB)以及天 门冬氨酸氨基转移酶(AST)的释放量的改变; 采用异硫氰酸荧光素-磷脂结合蛋白V/碘化丙啶 (FITC-AV/PI)双染色法检测前五组细胞凋亡率 的情况;采用蛋白质免疫印迹法(Western blot) 检测加入抑制剂LY294002 前后丝苏氨酸蛋白激 酶(Akt)、磷酸化丝苏氨酸蛋白激酶(p-Akt)、 B 细胞淋巴瘤/白血病-2 相关x 蛋白(Bax)、B 细胞淋巴瘤/白血病-2(Bcl-2)及半胱氨酸天冬氨 酸蛋白酶-9(caspase-9)等细胞作用信号通路 PI3K/Akt 相关蛋白的情况。结论( 1 ) Hoechst 33342 染色荧光显微镜显示 OGD+HA+GA 组抗凋亡作用最明显;(2)透射 电镜观察OGD+HA+GA 组凋亡现象改善最多; (3)LDH、CK-MB 及AST 的含量变化显示 OGD+HA+GA 组心肌细胞损伤指标降低最多 (P<0.05);(4)Western blot 法检测结果显示 HA+GA可以减少OGD对H9c2心肌细胞的损伤, 其作用机制与PI3K/Akt 信号通路有关。