Paeoniflorin Ameliorates Colonic Fibrosis in Rats with Postinfectious Irritable Bowel Syndrome by Inhibiting the Leptin/LepRb Pathway.

Postinfectious irritable bowel syndrome (PI-IBS) is a highly prevalent gastrointestinal disorder associated with immune dysregulation and depression- and anxiety-like behaviors. Through traditional medicine, the active ingredient of Paeoniae Radix called paeoniflorin (PF) was previously found to prevent the symptoms of PI-IBS. However, there is limited information on the effects of PF on intestinal function and depression- and anxiety-like symptoms in PI-IBS animal models. Here, we aimed to determine the effects of PF treatment on the symptoms of PI-IBS in a rat model. The PI-IBS rat model was established via early postnatal sibling deprivation (EPSD), trinitrobenzenesulfonic acid (TNBS), and chronic unpredictable mild stress (CUMS) stimulation and then treated with different dosages of PF (10, 20, and 40 mg/kg) and leptin (1 and 10 mg/kg). The fecal water content and body weight were measured to evaluate the intestinal function, while the two-bottle test for sucrose intake, open field test (OFT), and elevated plus maze test (EMT) were performed to assess behavioral changes. The serum leptin levels were also measured using an enzyme-linked immunosorbent assay. Furthermore, the expressions of leptin and its receptor, LepRb, were detected in colonic mucosal tissues through an immunohistochemical assay. The activation of the PI3K/AKT signaling pathway and the expression of brain-derived neurotrophic factor (BDNF) were also detected via western blotting. After the experimental period, the PI-IBS rats presented decreased body weight and increased fecal water content, which coincided with elevated leptin levels and heightened depression- and anxiety-like behaviors (e.g., low sucrose intake, less frequency in the center areas during OFT, and fewer activities in the open arms during EMT). However, the PF treatment ameliorated these observed symptoms. Furthermore, PF not only inhibited leptin/LepRb expression but also reduced the PI3K/AKT phosphorylation and BDNF expression in PI-IBS rats. Notably, cotreatment with leptin (10 mg/kg) reduced the effects of PF (20 mg/kg) on colonic fibrosis, leptin/LepRb expression, and PI3K/AKT activation. Therefore, our findings suggest that leptin is targeted by PF via the leptin/LepRb pathway, consequently ameliorating the symptoms of PI-IBS. Our study also contributes novel insights for elucidating the pharmacological action of PF on gastrointestinal disorders and may be used for the clinical treatment of PI-IBS in the future.

Suppression of PTRF Alleviates Post-Infectious Irritable Bowel Syndrome via Downregulation of the TLR4 Pathway in Rats.

Background: Accumulating evidence suggests that the polymerase I and transcript release factor (PTRF), a key component of the caveolae structure on the plasma membrane, plays a pivotal role in suppressing the progression of colorectal cancers. However, the role of PTRF in the development of functional gastrointestinal (GI) disorders remains unclear. Post-infectious irritable bowel syndrome (PI-IBS) is a common functional GI disorder that occurs after an acute GI infection. Here, we focused on the role of PTRF in the occurrence of PI-IBS and investigated the underlying mechanisms. Methods: Lipopolysaccharide (LPS) (5 µg/ml) was used to induce inflammatory injury in human primary colonic epithelial cells (HCoEpiCs). Furthermore, a rat model of PI-IBS was used to study the role of PTRF. Intestinal sensitivity was assessed based on the fecal water content. A two-bottle sucrose intake test was used to evaluate behavioral changes. Furthermore, shRNA-mediated knockdown of PTRF was performed both in vitro and in vivo. We detected the expression of PTRF in colonic mucosal tissues through immunohistochemistry (IHC), western blotting (WB), and immunofluorescence (IF) analysis. Luciferase activity was quantified using a luciferase assay. Co-localization of PTRF and Toll-like receptor 4 (TLR4) was detected using IF analysis. The activation of the signaling pathways downstream of TLR4, including the iNOs, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) pathways, was detected via WB. The levels of NO, IL-1ß, IL-6, and TNF-α were measured using enzyme-linked immunosorbent assays. Results: LPS significantly induced PTRF expression and signaling downstream of TLR4, including p38, ERK, and JNK pathways, in HCoEpiCs. Moreover, shRNA-mediated knockdown of PTRF in HCoEpiCs significantly decreased the phosphorylation of JNK, ERK, and p38 and iNOS expression. In PI-IBS rats, the lack of PTRF not only reduced fecal water content and suppressed depressive behavior but also increased the body weight. Furthermore, we found a strong co-localization pattern for PTRF and TLR4. Consistently, the lack of PTRF impaired TLR4 signaling, as shown by the decreased levels of p-JNK, p-ERK, and p-p38, which are upstream factors involved in iNOS expression. Conclusion: PTRF promoted PI-IBS and stimulated TLR4 signaling both in vitro and in vivo. The results of this study not only enlighten the pathogenesis of PI-IBS but also help us understand the biological activity of PTRF and provide an important basis for the clinical treatment of PI-IBS by targeting PTRF.

Effects of Jian Pi Qu Shi Formula on intestinal bacterial flora in patients with idiopathic membranous nephropathy: A prospective randomized controlled trial.

BACKGROUND: The incidence of idiopathic membranous nephropathy (IMN) has recently increased remarkably. Immune dysfunction caused by disordered intestinal flora might be an important factor affecting IMN. The Jian Pi Qu Shi Formula (JPQSF) shows promise in treating IMN. Here, we sequenced 16S rRNA genes to compare intestinal flora between patients with IMN and healthy persons. We also conducted a randomized controlled clinical trial to further compare the intestinal flora of patients with IMN treated with traditional Chinese medicine (TCM) and western medicine (WM). METHODS: Among 40 patients with IMN treated at Department of Nephrology in Xiyuan Hospital, Chinese Academy of Traditional Chinese Medicine between July 2016 and December 2018, we compared 30 of them with 10 healthy persons (controls). The IMN group was randomly assigned to receive JPQSF (TCM) or immunosuppressant WM therapy in (n = 15 per group) for 6 months. Intestinal microbiota diversity was analyzed using alpha diversity and beta diversity. Intestinal flora that significantly differed between the groups was analyzed using MetaStat. The effects and safety of the therapies were determined based on the values for plasma albumin, 24-h urine protein excretion, serum creatinine, urea nitrogen, estimate glomerular filtration rate (eGFR), complete blood count, and liver enzymes. All data were statistically analyzed using Statistical Package for the Social Sciences (SPSS) 20.0 statistical software. RESULTS: Baseline characteristics did not significantly differ between the IMN and healthy groups, or the TCM and WM groups. After six months of treatment, 24-h urinary protein significantly declined in the TCM and WM groups (before and after treatment: 3.24 ± 1.74 vs. 1.73 ± 1.85 g, P < 0.05 and 3.94 ± 1.05 vs. 1.91 ± 1.18 g, P < 0.05, respectively). Plasma albumin was significantly increased in the TCM group (before vs. after treatment: 32.44 ± 9.04 vs. 39.99 ± 7.03 g/L, P < 0.05), but did not significantly change in the WM group (31.55 ± 4.23 vs. 34.83 ± 9.14 g/L, P > 0.05). Values for urea nitrogen, serum creatinine, and eGFR did not significantly change in either group. The alpha diversity index for intestinal flora differed between the IMN and healthy groups, and the TCM and WM groups. Comparisons of multiple samples (beta diversity) revealed differences in intestinal flora between the IMN and healthy groups, and the TCM and WM groups. The Metastat analysis findings showed that the main genera that differed between the IMN group before treatment and the healthy group were Christensenellaceae_R-7_group, Bifidobacterium (77), Dorea, Escherichia-Shigella, Parabacteroides, Bifidobacterium, and Coprococcus_3. After TCM therapy, the main differential genera were Butyricimonas, Bacteroides, Alistipes, and Lachnospira, and after WM therapy, these were Ruminococcus_2, Lachnospiraceae_ND3007_group, Lachnospira, Bifidobacterium, Alistipes, and [Eubacterium]_ventriosum_group. CONCLUSION: Patients with IMN might have disordered intestinal flora, and JPQSF can regulate intestinal flora in patients with IMN.

Triptolide Attenuates Vascular Calcification by Upregulating Expression of miRNA-204.

Background: Triptolide (TP), a naturally derived compound from Tripterygium wilfordii, has been proven effective in protecting against cardiovascular system, but the molecular mechanisms underlying its protective effects are poorly understood. In the current study, we sought to test the potential protective role of TP in the regulation of vascular calcification in a rat model and explore whether TP attenuates medial vascular calcification by upregulating miRNA-204. Methods: Vitamin D3 plus nicotine (VDN) was used to induce a vascular calcification (VC) model of rat aorta. Von Kossa and Hematoxylin-Eosin staining were applied to assess the degree of calcification of rat aortas. Calcium content and alkaline phosphatase activity were measured. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was applied to quantify miRNA-204 expression. The localization of runt-related transcription factor-2 (RUNX2) and bone morphogenetic protein-2 (BMP2) expressions were detected by immunohistochemistry and western blotting. Results: Administration of TP greatly reduced vascular calcification in a dose-dependent manner compared with VC controls. The increase in ALP activity and calcium content was ameliorated by TP. Moreover, protein expression levels of BMP2 and RUNX2 were significantly reduced in calcified aortas. MiRNA-204 expression was increased in the TP-treated groups compared with VC controls and the effects of TP were reversed by the intravenous injection of miRNA-204-interfering lentivirus. However, the miRNA-204-overexpressing lentivirus had no additional effects on ALP activity, calcium content, BMP2 and RUNX2 expressions compared with those from TP group. Conclusion: TP inhibited BMP2 and RUNX2 expression and attenuated vascular calcification via upregulating the level of miRNA-204. TP appears to be a potential new therapeutic option for treating vascular calcification.

Surveilance situation of Salmonella in foodborne diseases in Henan,China 2015-2016 / 中国人兽共患病学报

We investigated the infection situation,serotype distribution,sources of etiological food and drug resistance of Salmonella in foodborne disease patients in Henan Province in 2015 and 2016.We evenly arranged 15 sentinel hospitals in Henan Province in 2015 and 2016,and a total of 5 720 patient defined cases were monitored,whose information was collected.A total of 221 Salmonella strains were isolated from the fecal of diarrhea patients,who were studied on serotyping,drug resistance and traceability of related etiological food,and the results were analyzed statistically.Results showed that the S.enteritidis,S.typhimurium and S.thompson were dominant types for serotyping in the 221 Salmonella strains,and 221 strains were widely distributed in 46 serotypes,the serotype distribution was more extensive;dairy and dairy products and meat and meat products were main suspicious etiological foods types caused by Salmonella.For drug susceptibility test of 11 kinds of antibiotics,the susceptibility of Salmonella to Cefoxitin,Cefotaxime,Chloramphenicol and Trimethoprim/Sulfamethoxazole significantly decreased (P＜0.05),and that to Ciprofloxacin,Ampicillin,Tetracycline and Ampicillin/Sulbactam decreased significantly (P＜0.01);only that to Ciprofloxacin,Nalidixic acid and Gentamicin decreased insignificantly (P＜0.05).Relevant departments should strengthen the meat and meat products market supervision,to make great efforts for control the use of antibiotics,strengthen the active surveillance of Salmonella disease and drug resistance,and to reduce the incidence of foodborne diseases.

Herbal prescription Chang'an II repairs intestinal mucosal barrier in rats with post-inflammation irritable bowel syndrome.

AIM: The herbal prescription Chang'an II is derived from a classical TCM formula Tong-Xie-Yao-Fang for the treatment of liver-qi stagnation and spleen deficiency syndrome of irritable bowel syndrome (IBS). In this study we investigated the effects of Chang'an II on the intestinal mucosal immune barrier in a rat post-inflammation IBS (PI-IBS) model. METHODS: A rat model of PI-IBS was established using a multi-stimulation paradigm including early postnatal sibling deprivation, bondage and intrarectal administration of TNBS. Four weeks after TNBS administration, the rats were treated with Chang'an II (2.85, 5.71 and 11.42 g · kg(-1) · d(-1), ig) for 14 d. Intestinal sensitivity was assessed based on the abdominal withdrawal reflex (AWR) scores and fecal water content. Open field test and two-bottle sucrose intake test were used to evaluate the behavioral changes. CD4(+) and CD8(+) cells were counted and IL-1ß and IL-4 levels were measured in intestinal mucosa. Transmission electron microscopy was used to evaluate ultrastructural changes of the intestinal mucosal barrier. RESULTS: PI-IBS model rats showed significantly increased AWR reactivity and fecal water content, and decreased locomotor activity and sucrose intake. Chang'an II treatment not only reduced AWR reactivity and fecal water content, but also suppressed the anxiety and depressive behaviors. Ultrastructural study revealed that the gut mucosal barrier function was severely damaged in PI-IBS model rats, whereas Chang'an II treatment relieved intestinal mucosal inflammation and repaired the gut mucosal barrier. Furthermore, PI-IBS model rats showed a significantly reduced CD4(+)/CD8(+) cell ratio in lamina propria and submucosa, and increased IL-1ß and reduced IL-4 expression in intestinal mucosa, whereas Chang'an II treatment reversed PI-IBS-induced changes in CD4(+)/CD8(+) cell ratio and expression of IL-1ß and IL-4. CONCLUSION: Chang'an II treatment protects the intestinal mucosa against PI-IBS through anti-inflammatory, immunomodulatory and anti-anxiety effects.

Ginsenoside Rd promotes neurogenesis in rat brain after transient focal cerebral ischemia via activation of PI3K/Akt pathway.

AIM: To investigate the effects of ginsenoside Rd (Rd) on neurogenesis in rat brain after ischemia/reperfusion injury (IRI). METHODS: Male SD rats were subjected to transient middle cerebral artery occlusion (MCAO) followed by reperfusion. The rats were injected with Rd (1, 2.5, and 5 mg·kg(-1)·d(-1), ip) from d 1 to d 3 after MCAO, and with BrdU (50 mg·kg(-1)·d(-1), ip) from d 3 to d 6, then sacrificed on 7 d. The infarct size and neurological scores were assessed. Neurogenesis in the brains was detected by BrdU, DCX, Nestin, and GFAP immunohistochemistry staining. PC12 cells subjected to OGD/reperfusion were used as an in vitro model of brain ischemia. VEGF and BDNF levels were assessed with ELISA, and Akt and ERK phosphorylation was measured using Western blotting. RESULTS: Rd administration dose-dependently decreased the infarct size and neurological scores in the rats with IRI. The high dose of Rd 5 (mg·kg(-1)·d(-1)) significantly increased Akt phosphorylation in ipsilateral hemisphere, and markedly increased the number of BrdU/DCX and Nestin/GFAP double-positive cells in ischemic area, which was partially blocked by co-administration of the PI3 kinase inhibitor LY294002. Treatment with Rd (25, 50, and 100 µmol/L) during reperfusion significantly increased the expression of VEGF and BDNF in PC12 cells with IRI. Furthermore, treatment with Rd dose-dependently increased the phosphorylation of Akt and ERK, and significantly decreased PC12 cell apoptosis, which were blocked by co-application of LY294002. CONCLUSION: Rd not only attenuates ischemia/reperfusion injury in rat brain, but also promotes neurogenesis via increasing VEGF and BDNF expression and activating the PI3K/Akt and ERK1/2 pathways.

Protective effect of kaempferol on LPS plus ATP-induced inflammatory response in cardiac fibroblasts.

Inflammatory response is an important mechanism in the pathogenesis of cardiovascular diseases. Cardiac fibroblasts play a crucial role in cardiac inflammation and might become a potential therapeutic target in cardiovascular diseases. Kaempferol, a flavonoid commonly existing in many edible fruits, vegetables, and Chinese herbs, is well known to possess anti-inflammatory property and thus has a therapeutic potential for the treatment of inflammatory diseases. To date, the effect of kaempferol on cardiac fibroblasts inflammation is unknown. In this study, we investigated the anti-inflammatory effect of kaempferol on lipopolysaccharide (LPS) plus ATP-induced cardiac fibroblasts and explored the underlying mechanisms. Our results showed that kaempferol at concentrations of 12.5 and 25 µg/mL significantly suppressed the release of TNF-α, IL-1ß, IL-6, and IL-18 and inhibited activation of NF-κB and Akt in LPS plus ATP-induced cardiac fibroblasts. These findings suggest that kaempferol attenuates cardiac fibroblast inflammation through suppression of activation of NF-κB and Akt.

[Protective effect of rosmarinic acid on hypoxia/reoxygenation injury in cardiomyocytes].

OBJECTIVE: To study the protective effect of rosmarinic acid (Ros A) on the primary cardiomyocyte hypoxia/reoxygenation (H/R) injury. METHOD: Primary cardiomyocytes of rats were cultured in vitro to establish the H/R injury of cardiomyocytes and observe the changes in the cell viability and LDH leakage. The changes in ATP content and ROS in cardiomyocytes were measured by using chemiluminescence and fluorescent probe technique. The effects of rosmarinic acid on the apoptosis of cardiomyocytes, cleaved-caspase 3, Akt and p-Akt protein expression were further detected by flow cytometry and western blot analysis. RESULT: According to the experimental results, Ros A at doses of 25, 50, 100 mg x L(-1) could inhibit the decrease in H/R-induced cell viability, LDH leakage and excessive ROS generation, and maintain the ATP level in cells. Ros A at doses of 50, 100 mg x L(-1) could remarkably inhibit the H/R-induced cardiomyocyte apoptosis and down-regulate the expression of cleaved caspase-3. Moreover, Ros A at doses of 100 mg x L(-1) could significantly up-regulate the expression of p-Akt. CONCLUSION: Ros A has the significant effect in resisting the cardiomyocyte H/R injury, improve cardiomyocyte energy metabolism and reduce cell apoptosis, which is related to the activation of Akt pathway.

[Study on effect of huatuo zaizao extractum on focal cerebral ischemia/reperfusion neurogenesis in rats and its mechanisms].

OBJECTIVE: To observe the effect of Huatuo Zaizao extractum (HTZZ) on focal cerebral ischemia/reperfusion (I/R) neurogenesis in rats induced by middle cerebral artery occlusion (MCAO) and its mechanism. METHOD: Totally 55 healthy adult male Sprague-Dawley rats were divided into the sham operation group, the MCAO model group and HTZZ high, middle and low dose groups (5, 2.5, 1.25 g x kg(-1)), with 11 rats in each group, and orally administered with drugs. The focal cerebral ischemia model was established by performing a middle cerebral artery occlusion (MCAO, 90 min) followed by a seven-day reperfusion (once a day). The neurogenesis and expressions of extracellular signal-regulated kinase (ERK) and cAMP response element binding protein (CREB) were detected by the immunofluorescent staining. The enzyme linked immunosorbent assay (ELISA) was adopted to determine the vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). RESULT: MCAO (90 min) followed by a seven-day reperfusion resulted in the significant increase in the number of penumbra cortex newborn neurons (BrdU(+) -NeuN(+)), which was accompanied by the growth of ERK and CREB phosphorylation and VEGF and BDNF levels. HTZZ could promote the generation of newborn neurons (BrdU(+)-NeuN(+)) and the ERK and CREB phosphorylation and increase VEGF and BDNF levels at the ischemic side. CONCLUSION: HTZZ could promote the neurogenesis, which may be the interventional targets of effective traditional Chinese medicine Huatuo Zaizao extractum in promoting the self-repair function of the cerebral ischemic areas.

[Intervention effect of quercetin on inflammatory secretion of cardiac fibroblasts].

To establish neonatal rat cardiac fibroblast inflammatory secretion model by using LPS 100 microg x L(-1) combined with ATP 5 mmol x L(-1), in order to study the inhibitory effect of quercetin on the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts, further investigate the effect of quercetin on the protein expression of p-NF-kappaB p65 (S276) and p-Akt (S473) by western blot, and discuss the inhibitory effect of quercetin on the inflammatory secretion of cardiac fibroblasts. According to the findings, quercetin with the concentrations between 51.74 micromol x L(-1) and 827.81 micromol x L(-1) had no significant effect on the activity of cardiac fibroblasts. Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of TNF-alpha and IL-1beta induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.01 or P < 0.05). Quercetin with the concentrations of 82.78, 41.39 micromol x L(-1) could notably inhibit the increase of IL-6 induced LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.05), without any notable effect of quercetin with the concentration of 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20. 70 micromol x L(-1) could notably inhibit the NF-kappaB p65 (S276) activation induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 15 min, with the most significant effect in 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of p-Akt(473) expression induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 240 min (P < 0.05). Therefore, this study believes that quercetin could attenuate the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts by inhibiting the activation of NF-kappaB p65 (S276) and Akt (473).

Cardioprotective effect of protocatechuic acid on myocardial ischemia/reperfusion injury.

Protocatechuic acid (PCA), a phenolic compound and one of the main metabolites of complex polyphenols, has been found to possess various biological activities, and it may have a potential in the treatment of ischemic heart diseases. This study explored the cardioprotective effect of PCA on myocardial ischemia/reperfusion (MI/R) injury and the underlying mechanisms. In an in vivo rat model of MI/R injury, myocardial infarct size, serum TNF-a level, and platelet aggregation were measured. In a primary neonatal rat cardiomyocyte model of hypoxia/ reoxygenation (H/R) injury, the apoptotic rate, expressions of cleaved caspase-3, and phosphorylated Akt were observed. We found that PCA significantly reduced myocardial infarct size, serum TNF-a level, and platelet aggregation. In vitro experiments revealed that PCA significantly inhibited the apoptotic rate and the expression of cleaved caspase-3, and it upregulated the expression of phosphorylated Akt in cardiomyocytes subjected to H/R injury. Our results suggest that PCA can provide a significant protection against MI/R injury, which may be at least partially attributed to its inhibitions against injury induced by MI/R including the inflammatory response, platelet aggregation, and cardiomyocytes apoptosis.

Total glucosides of paeony inhibit the proliferation of fibroblast-like synoviocytes through the regulation of G proteins in rats with collagen-induced arthritis.

The aim of this study was to investigate the expression of G proteins in fibroblast-like synoviocytes (FLSs) from rats with collagen-induced arthritis (CIA) and to determine the effect of total glucosides of paeony (TGP). CIA rats were induced with chicken type II collagen (CCII) in Freund's complete adjuvant. The rats with experimental arthritis were randomly separated into five groups and then treated with TGP (25, 50, and 100mg/kg) from days 14 to 35 after immunization. The secondary inflammatory reactions were evaluated through the polyarthritis index and histopathological changes. The level of cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. The FLS proliferation response was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The toxin-catalyzed ADP-ribosylation of G proteins was performed through autoradiography. The results show that TGP (25, 50, and 100mg/kg) significantly decreased the arthritis scores of CIA rats and improved the histopathological changes. TGP inhibited the proliferation of FLSs and increased the level of cAMP. Moreover, the FLS proliferation and the level of Gαi expression were significantly increased, but the level of Gαs expression was decreased after stimulation with IL-1ß (10ng/ml) in vitro. TGP (12.5 and 62.5µg/ml) significantly inhibited the FLS proliferation and regulated the balance between Gαi and Gαs. These results demonstrate that TGP may exert its anti-inflammatory effects through the suppression of FLS proliferation, which may be associated with its ability to regulate the balance of G proteins. Thus, TGP may have potential as a therapeutic agent for the treatment of rheumatoid arthritis.

[Study on effect of weinaokang and bilobalide on autophagy and neurogenesis induced by focal cerebral ischemia reperfusion].

OBJECTIVE: To observe the protective effect of the Weinaokang (WNK) and its active compound bilobalide on focal cerebral ischemia reperfusion, and their mechanisms. METHOD: The 60-minute middle cerebral artery occlusion (MCAO) was adopted to establish the 24 h-14 d reperfusion model. The expression of Beclin-1 was detected by the Western blotting technique. The transmission electron microscopy was used to observe ultrastructural changes. Neurogenesis was detected by the immunofluorescence staining. RESULT: WNK (20, 10 mg x kg(-1), ig) or its active compound bilobalide (10, 5 mg x kg(-1), ig) could promote the generation of mature neurons (BrdU(+) -MAP-2+) at the ischemic side, and inhibit expression of autophagy-related gene Beclin-1, so as to reduce the neuron injury induced by focal cerebral ischemia reperfusion. CONCLUSION: WNK and its active compound bilobalide can inhibit neuron autophagy and improve neurogenesis in ischemic peripheral area, suggesting that neurogenesis may be the intervention target for WNK to promote self-repairing of ischemic area.

[Effect and mechanism of huatuo zaizao extractum on focal cerebral ischemia/reperfusion-induced blood-brain barrier injury in rats].

OBJECTIVE: To observe the effect and mechanism of Huatuo Zaizao extractum (HTZZ) on focal ischemia/reperfusion (I/R) blood-brain barrier injury induced by middle cerebral artery occlusion. METHOD: Sixty healthy male adult Sprague-Dawley rats was randomly divided into the sham operation group, the MCAO model group, the Tanakan (20 mg x kg(-1)) group, and high, middle and low-dose HTZZ groups (5, 2.5, 1.25 g x kg(-1)), with 10 in each group and single-dose duodenal administration. Middle cerebral artery occlusion was adopted to establish the rat focal I/R model. After ischemia for 90 min and reperfusion for 24 h, the pathological injury at the ischemia side was observed by HE staining. The blood-brain barrier structure was observed under transmission electron microscope. Expressions of G protein-coupled receptor kinases 2 (GRK2), matrix metalloproteinases 2 (MMP-2) and MMP-9 were detected by western blotting technique. RESULT: After 90 min MCAO/24 h reperfusion, penumbra cerebral cortical micro-vessels showed edema, mitochondrial injury, vacuolation, membrane injury and reduction. Along with the changes, sub-cells of G protein-coupled receptor kinase 2 (GRK2) in cortical penumbra brain tissues transferred from cytoplasm to membrane, with increase in expressions of MMP-2 and MMP-9. HTZZ could effectively recover cerebral micro-vascular endothelial edemaand blood-brain barrier ultrastructure injury induced by I/R, reduce expression of functional (membrane coupling) GRK2, and inhibit expressions of MMP-2 and MMP-9. CONCLUSION: Cell membrane coupling GRK2 may be the effective target of Huatuo Zaizao extractum.

[Protective effect of succinic acid on primary cardiomyocyte hypoxia/reoxygenation injury].

To establish cardiomyocyte hypoxia/reoxygenation injury model by culturing primary cardiomyocytes from suckling SD rats, in order to study the effect of succinic acid on LDH leakage rate cardiomyocyte ischemia/reperfusion injury. Furthermore, flow cytometry and western blot were conducted to detect the effect of succinic acid on cardiomyocyte apoptosis, cleaved caspase-3 and p-Akt, and discuss the protective effect of succinic acid on primary cardiomyocyte hypoxia/reoxygenation injury of primary cardiomyocytes from neonatal SD rats. According to the findings of the study, succinic acid at the concentrations ranging between 31.25 mg x L(-1) and 500 mg x L(-1) had no significant effect on primary cardiomyocyte activity, and succinic acid at the concentrations of 400, 200, 100, 50 mg x L(-1) could notably reduce cardiomyocyte ischemia/reperfusion LDH leakage rate (P < 0.01 or P < 0.05, respectively). Succinic acid at the concentrations of 400 mg x L(-1) and 200 mg x L(-1) could significantly reduce the percentage of cardiomyocyte apoptosis (P < 0.05), and inhibit the protein expression of cleaved caspase-3 caused by cardiomyocyte ischemia/reperfusion (P < 0.05). Succinic acid at the concentration of 400 mg x L(-1) could remarkably increase the protein expression of cardiomyocyte Akt (P < 0.05), while succinic acid at the concentration of 200 mg x L(-1) had no obvious effect on the protein expression of cardiomyocyte Akt. Therefore, this study demonstrated that succinic acid could inhibit necrosis and apoptosis caused by cardiomyocyte hypoxia/reoxygenation by activating Akt phosphorylation.

Hawthorn leaves flavonoids decreases inflammation related to acute myocardial ischemia/reperfusion in anesthetized dogs.

OBJECTIVE: To investigate the effects and mechanisms of hawthorn leaves flavonoids (HLF) on acute myocardial ischemia/reperfusion in anesthetized dogs. METHODS: The acute ischemia models were prepared by ligating left anterior descending (LAD) artery for 60 min. Qualified 15 male dogs were randomly divided into 3 groups with 5 in each group: blank control (treated with normal saline 3 mL/kg) group, HLF low dosage (5 mg/kg) group and high dosage (10 mg/kg) group, with an once injection through a femoral vein 5 min before reperfusion. Epicardial electrocardiogram was adopted to measure the scope and degree of myocardial ischemia. Simultaneously, neutrophil infiltration in infarct (Inf) and remote site (RS) of myocardial tissue was measured by myeloperoxidase (MPO) activity assay. The serum interleukin-1 (IL-1) and tumor necrosis factorα (TNF-α) content were quantified by radioimmuno-assay. Furthermore, expression of G protein-coupled receptor kinase 2 (GRK2) and nuclear factor κB (NF-κB) in Inf and RS tissue were detected by Western blotting technique. RESULTS: Ischemia and reperfusion increased the MPO activity and IL-1 and TNF-α content. HLF (10 and 5 mg/kg) could significantly decrease the degree and scope of myocardial ischemia; markedly inhibit the increase of MPO activity, and IL-1 and TNF-α content induced by myocardial ischemia/infarction. Furthermore, HLF increased GRK2 expression and inhibited NF-κB expression in Inf tissue. CONCLUSION: HLF could improve the situation of acute myocardial ischemia and inhibit the inflammation in anesthetized dogs, which might be due to its increasing effect on the GRK2 and NF-κB expressions.

Effects and mechanism of Weinaokang on reperfusion-induced vascular injury to cerebral microvessels after global cerebral ischemia.

OBJECTIVE: To study the effects of the Weinaokang (WNK), the active compounds extracted from Ginkgo, Ginseng, and saffron, on ischemia/reperfusion (I/R)-induced vascular injury to cerebral microvessels after global cerebral ischemia. METHODS: Male C57BL/6J mice were randomly divided into 5 groups (10 animals/group): the sham group (0.5% CMC-Na, 20 mL/kg), the I/R model group (0.5% CMCNa, 20 mL/kg), the I/R+Crocin control group (20 mg/kg), the I/R+high dose WNK group (20 mg/kg), and the I/R+low dose WNK group (10 mg/kg). Bilateral common carotid artery occlusion (BCCAO, 20 min) in mice, followed by 24 h reperfusion, was built. The generation of nitric oxide (NO), the activity of nitric oxide synthase (NOS), the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), and the expression of matrix metalloproteinases-9 (MMP-9) and G protein-coupled receptor kinase 2 (GRK2) in cortical microvascular homogenates were evaluated. The ultrastructural morphology of cortical microvascular endothelial cells (CMEC) was observed. RESULTS: The transient global cerebral ischemia (20 min), followed by 24 h of reperfusion, significantly promoted the generation of NO and the activity of NOS. The reperfusion led to serious edema with mitochondrial injuries in the cortical CMEC, as well as enhanced membrane GRK2 expression and reduced cytosol GRK2 expression. Furthermore, enhanced phosphorylation of ERK1/2 and decreased expression of MMP-9 were detected in cortical microvessels after I/R (20 min/24 h). As well as the positive control Crocin (20 mg/kg, 21days), pre-treatment with WNK (20, 10 mg/kg, 21 days) markedly inhibited nitrative injury and modulated the ultrastructure of CMEC. Furthermore, WNK inhibited GRK2 translocation from cytosol to the membrane (at 20 mg/kg) and reduced ERK1/2 phosphorylation and MMP-9 expression in cortical microvessels. CONCLUSION: WNK and its active compounds (Crocin) are effective to suppress I/R-induced vascular injury to cerebral microvessels after global cerebral ischemia with the target on GRK2 pathways.

RNA interference-mediated downregulation of Beclin1 attenuates cerebral ischemic injury in rats.

AIM: To test the role of the Beclin 1-dependent autophagy pathway in brain damage during cerebral ischemia. METHODS: Focal cerebral ischemia was established in rats using a middle cerebral artery occlusion (MCAO) model. A lentiviral vector-associated RNA interference (RNAi) system was stereotaxically injected into the ipsilateral lateral ventricle to reduce Beclin1 expression. We measured the ipsilateral infarct volume, autophagosome formation, neurogenesis and apoptosis, all of which could be modulated by Beclin1 RNAi. RESULTS: On the 14th day after MCAO, Beclin1 downregulation by RNAi increased the population of neural progenitor cells (BrdU(+)-DCX(+)), newborn immature cells (BrdU(+)-Tuj-1(+)) and mature neurons (BrdU(+)-MAP-2(+)), and reduced the apoptosis of immature neurons (caspase-3(+)-DCX(+) and caspase-3(+)-Tuj-1(+)) surrounding the ischemic core of the ipsilateral hemisphere. Furthermore, RNAi-mediated downregulation of Beclin1 decreased infarct volume and inhibited histological injury and neurological deficits. CONCLUSION: RNAi-mediated downregulation of Beclin1 improves outcomes after transient MCAO.Acta Pharmacologica Sinica (2009) 30: 919-927; doi: 10.1038/aps.2009.79.

Effects of early postnatal sibling deprivation on anxiety and vulnerability to cocaine in offspring rats.

RATIONALE: Early-life experience has long-term consequences on affective behavior and drug abuse in adults. While many manipulations used to study these consequences alter mother-infant interactions, the effects of sibling interactions are less well characterized. OBJECTIVES: To examine the long-term effects of early postnatal sibling deprivation (EPSD) on anxiety-like behavior, sucrose preference and behavioral responses to cocaine in adult rats. MATERIALS AND METHODS: After EPSD manipulation, in which litters were culled to one pup on postnatal day 1 (PN1) or 7 (PN7), the dams' maternal behavior was observed. After the pups reached adulthood, we tested their behavioral responses in the elevated plus maze and sucrose consumption, and to cocaine conditioned place preference and cocaine sensitization. RESULTS: The pups with EPSD on PN1 received more maternal licking/grooming during the first postnatal week. EPSD on PN1 but not PN7 enhanced locomotor activity in the open field test and exploration of open arms in the elevated plus maze in both female and male offspring. While EPSD had no effect on sucrose intake in adult rats, it decreased vulnerability to cocaine sensitization and cocaine conditioned place preference in male but not female rats. CONCLUSION: Our findings that early postnatal sibling deprivation influences maternal licking/grooming behavior, as well as anxiety-like behavior and vulnerability to drugs in pups that have grown to adulthood, suggests that both sibling interaction and maternal behavior, play critical roles in individual development.