Protection by 3'-methoxypuerarin of rat hippocampal neurons against ischemia/reperfusion injury

3'-Methoxypuerarin (3'-MOP) is an isoflavone extracted from radix puerariae. The aim of this study was to investigate the role and the mechanism of 3'-MOP in the protection of hippocampal neurons against cerebral ischemia/reperfusion (I/R) injury in rats. I/R injury was induced by a modified four-vessel occlusion model. Rats were randomly divided into an I/R group, an I/R + 3'-MOP group and a control group. Histological changes in the neurons of the hippocampal CA1 region were observed with hematoxylin and eosine (H&E) staining. The apoptotic neurons in the hippocampal CA1 area were counted with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The results showed that compared with the I/R group, 3'-MOP increased the number of surviving neurons in the hippocampal CA1 region (P < 0.001) and markedly reduced the number of apoptotic pyramidal neurons (P < 0.001) after I/R injury. In conclusion, 3'-MOP can protect hippocampal neurons against I/R injury by inhibiting apoptosis.     

生长素对胃运动和对海马胃肠敏感神经元放电活动的影响

目的:探讨海马ghrelin对GD敏感神经元放电和弓状核ghrelin对胃运动的影响。方法:在细胞外记录海马的放电情况,并且检测清醒大鼠的胃运动。通过PCR免疫印迹和免疫荧光组织化学染色等方法来测定GHSR-1a在海马中的表达。用逆行追踪和免疫荧光组织化学染色检测ghrelin神经元的投射情况。Ghrelin况荧光金双标记的神经元以及GHSR-1a的表达分别可以在ARC和海马中观察到。结果:Ghrelin或者ARC电刺激可以兴奋海马区的胃牵张敏感神经元。Ghrelin受体拮抗剂[d-Lys-3]-GHRP-6预处理可以完全或者部分阻断这种兴奋作用。海马注射ghrelin可以显著促进胃运动,并且呈现剂量依赖关系,而且这种作用可以被[d-Lys-3]-GHRP-6所阻断。电刺激ARC能够促进胃运动。然而,预处理时[d-Lys-3]-GHRP-6可以减弱这些作用。电损毁海马可以减弱胃运动的兴奋作用,这个作用通过电刺激ARC产生的。结论:通过海马促进胃运动中ghrelin起着重要的作用。ARC可能参与调节海马对胃动力的影响。     

Orexigenic hormone ghrelin attenuates local and remote organ injury after intestinal ischemia-reperfusion

Background

Gut ischemia/reperfusion (I/R) injury is a serious condition in intensive care patients. Activation of immune cells adjacent to the huge endothelial cell surface area of the intestinal microvasculature produces initially local and then systemic inflammatory responses. Stimulation of the vagus nerve can rapidly attenuate systemic inflammatory responses through inhibiting the activation of macrophages and endothelial cells. Ghrelin, a novel orexigenic hormone, is produced predominately in the gastrointestinal system. Ghrelin receptors are expressed at a high density in the dorsal vagal complex of the brain stem. In this study, we investigated the regulation of the cholinergic anti-inflammatory pathway by the novel gastrointestinal hormone, ghrelin, after gut I/R.

Methods and Findings

Gut ischemia was induced by placing a microvascular clip across the superior mesenteric artery for 90 min in male adult rats. Our results showed that ghrelin levels were significantly reduced after gut I/R and that ghrelin administration inhibited pro-inflammatory cytokine release, reduced neutrophil infiltration, ameliorated intestinal barrier dysfunction, attenuated organ injury, and improved survival after gut I/R. Administration of a specific ghrelin receptor antagonist worsened gut I/R-induced organ injury and mortality. To determine whether ghrelin''s beneficial effects after gut I/R require the intact vagus nerve, vagotomy was performed in sham and gut I/R animals immediately prior to the induction of gut ischemia. Our result showed that vagotomy completely eliminated ghrelin''s beneficial effect after gut I/R. To further confirm that ghrelin''s beneficial effects after gut I/R are mediated through the central nervous system, intracerebroventricular administration of ghrelin was performed at the beginning of reperfusion after 90-min gut ischemia. Our result showed that intracerebroventricular injection of ghrelin also protected the rats from gut I/R injury.

Conclusions

These findings suggest that ghrelin attenuates excessive inflammation and reduces organ injury after gut I/R through activation of the cholinergic anti-inflammatory pathway.     

Daphnetin protects hippocampal neurons from oxygen-glucose deprivation–induced injury

Daphnetin, a coumarin derivative extracted from Daphne odora var., was reported to possess a neuroprotective effect. Recently, it has been demonstrated that daphnetin attenuates ischemia/reperfusion (I/R) injury. However, the role of daphnetin in cerebral I/R injury and the potential mechanism have not been fully understood. The present study aimed to explore the regulatory roles of daphnetin on oxygen-glucose deprivation/reoxygenation (OGD/R)–induced cell injury in a model of hippocampal neurons. Our results demonstrated that daphnetin improved cell viability and reduced the lactate dehydrogenase leakage in OGD/R–stimulated hippocampal neurons. In addition, daphnetin inhibited oxidative stress and cell apoptosis in hippocampal neurons after OGD/R stimulation. Furthermore, daphnetin significantly enhanced the nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in hippocampal neurons exposed to OGD/R. Knockdown of Nrf2 blocked the protective effect of daphnetin on OGD/R–induced hippocampal neurons. In conclusion, these findings demonstrated that daphnetin attenuated oxidative stress and neuronal apoptosis after OGD/R injury through the activation of the Nrf2/HO-1 signaling pathway in hippocampal neurons. Thus, daphnetin may be a novel therapeutic agent for cerebral I/R injury.     

The effect of ghrelin pretreatment on epididymal sperm quality and tissue antioxidant enzyme activities after testicular ischemia/reperfusion in rats

Ghrelin, the endogenous ligand for growth hormone secretagogue receptor, has been reported to prevent ischemia/reperfusion (I/R) injury in various tissues by its antioxidant activity. Therefore, this study was aimed to investigate the effect of ghrelin on sperm quality and antioxidant enzyme activity in a rat testicular ischemia/reperfusion injury model. Forty-two male Wistar rats were divided into groups control, I/R, and I/R plus ghrelin. The right testes were rotated 720° for 1 h and were allowed to reperfuse for 4 h and 30 days thereafter. Ghrelin (40 nmol/kg IP) or vehicle (physiological saline) was administrated 15 min before reperfusion. After 4 h of reperfusion, a right orchiectomy was performed to measure the biochemical parameters. In addition, the sperm was collected from the epididymis after 30 days of reperfusion, and sperm characteristics were examined. The malondialdehyde levels of the testis tissues were significantly increased, but a statistically significant decrease was found in the superoxide dismutase, glutathione peroxidase, and catalase activities in the I/R group as compared with the control, indicating I/R injury. The sperm evaluation showed a significant reduction in all characteristics resulted from I/R compared with the control. In the ghrelin-treated group, the malondialdehyde values were significantly lowered, and only enzyme activity of glutathione peroxidase showed significant increases compared with the I/R group. Ghrelin significantly enhanced sperm motility, movement, and concentration but did not prevent I/R-induced reduction in membrane integrity in the testes of rats compared to the I/R group. Our results suggest that ghrelin treatment has a protective role on IR-induced testicular injury, and this effect may be due to its antioxidant properties.     

Ghrelin对糖尿病大鼠下丘脑弓状核胃扩张敏感神经元和胃运动的影响

目的:探讨Ghrelin对糖尿病大鼠下丘脑弓状核胃扩张敏感神经元和胃运动的影响。方法:逆行追踪结合免疫组化观察ARC中GHSR-1的表达,细胞外放电记录,观察ghrelin对GD神经元放电活动的影响及电刺激ARC对GD神经元放电活动和胃运动的影响。结果:电生理实验结果表明,在ARC Ghrelin能够能激发GD兴奋性神经元(GD-E)和GD抑制性神经元(GD-I)。然而,ghrelin可以兴奋更少的GD-E神经元,在正常大鼠中ghrelin对于GD-E的兴奋作用比在DM大鼠中的作用弱。在体胃运动研究表明,在ARC中微量注射ghrelin可以明显的增强胃运动,并且呈现剂量依赖关系。Ghrelin在糖尿病大鼠促胃动力作用低于正常大鼠。Ghrelin诱导的效应可被生长激素促分泌素受体(GHSR)拮抗剂阻断[d-lys-3]-GHRP-6或bim28163。放射免疫法和实时荧光定量PCR数据表明胃血浆ghrelin水平,在ARC ghrelin mRNA的表达水平先上升后下降,糖尿病大鼠(DM)中,在ARC中GHSR-1a mRNA表达保持在一个比较低的水平。结论:ghrelin可以调节GD敏感神经元以及胃运动,通过ARC中ghrelin受体。在糖尿病大鼠中,Ghrelin促进胃运动作用减弱可能与ARC中ghrelin受体表达减少有关。     

Effects of ghrelin on neuronal activity in the ventromedial nucleus of the hypothalamus in infantile rats: An in vitro study

Ghrelin is an endogenous ligand for the growth hormone (GH) secretagogue (GHS) receptor (GHS-R) and a potent stimulant for GH secretion even in infantile rats before puberty. Although the ventromedial nucleus of the hypothalamus (VMH) might be a site of action for ghrelin to induce GH release, the electrophysiological effect of ghrelin on VMH neurons in infantile rats remains to be elucidated. Thus, the purpose of the present study was to investigate the effect of ghrelin on VMH neurons using hypothalamic slices of infantile rats. Ghrelin excited a majority of VMH neurons in a concentration-dependent manner. VMH neurons that were excited by GH releasing peptide-6 (GHRP-6), a synthetic GHS, were also excited by ghrelin and vice versa. Repeated application of ghrelin to the same VMH neuron decreased progressively the excitatory responses depending on the number of times it was administered. The excitatory effect of ghrelin on VMH neurons in normal artificial cerebrospinal fluid (ACSF) persisted in low Ca²⁺-high Mg²⁺ ACSF. The present results indicate that (1) ghrelin excites a majority of VMH neurons dose-dependently and postsynaptically and (2) the excitatory effects of ghrelin are mimicked by GHRP-6 and desensitized by repeated applications of ghrelin.     

Dexmedetomidine alleviates cerebral ischemia-reperfusion injury in rats via inhibition of hypoxia-inducible factor-1α

Dexmedetomidine (Dex) was reported to reduce ischemia-reperfusion (I/R) injury in kidney and brain tissues. Thus, we aimed to study the role and mechanism of Dex in cerebral I/R injury by inhibiting hypoxia-inducible factor-1α (HIF-1α) and apoptosis. First, I/R injury models were established. Six groups were assigned after different treatments: sham, I/R, I/R+Dex, I/R+2-methoxyestradiol (2ME2) (HIF-1α inhibitor), I/R+CoCl ₂ (HIF-1α activator), and I/R+Dex+CoCl ₂ groups. Neurological function, cerebral infarction volume, survival, and apoptosis of brain cells were then analyzed. Besides, immunohistochemistry and Western blot analysis were used to detect the expression of HIF-1α, BCL-2[B-cell leukemia/lymphoma 2] adenovirus E1B interacting protein 3 (BNIP3), B-cell leukemia/lymphoma 2 (BCL2), BCL2[B-cell leukemia/lymphoma 2] associated X (Bax), and cleaved-caspase3 proteins in brain tissues. I/R rats showed cerebral infarction, increased neurological function score, number of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL)-positive cells and HIF-1α–positive cells as well as decreased neurons. Inhibition of HIF-1α can reduce the apoptosis induced by I/R, and overexpression of HIF-1α can aggravate apoptosis in brain tissue of I/R rats. Furthermore, activation of HIF-1α expression blocks the inhibitory effect of Dex on neuronal apoptosis in I/R rats. Dex may inhibit the neuronal apoptosis of I/R rats by inhibiting the HIF-1α pathway and then improve the cerebral I/R injury in rats.     

Effects of peripherally and centrally applied ghrelin in the pathogenesis of ischemia-reperfusion induced injury of the small intestine

Ghrelin is an important hormone involved in the control of the human appetite center. Recently, protective properties of this hormone have been recognized in various models of impairment of the gastric mucosa, including stress, ischemia and reperfusion (I/R). Ghrelin is predominantly secreted by the gastric mucosa of stomach, but there are other sources of ghrelin, for example in the hypothalamus and various parts of the central nervous system (CNS) that should be taken into consideration. This hormone exerts biological effects via the activation of growth hormone secretagogue receptor (GHSR), the presence of which was confirmed in different parts of the gastrointestinal (GI) tract and midbrain structures. Although substantial evidence of the divergent biological effects of ghrelin and the mechanism of its action has been emphasized, the precise mechanisms of ghrelin which affords GI protection is still unclear. Particularly, there is a sparse amount of evidence concerning its action on the GI system. The major aim of the present study was to evaluate the importance of peripherally and centrally administered ghrelin at different times of the ischemia and reperfusion (I/R period in the modulation of resistance of the intestinal mucosa to the injury induced by ischemia and subsequent reperfusion. Secondly, we wanted to evaluate the possible mechanism of the action of ghrelin with a particular focus on its influence on the intestinal blood flow. Male Wistar rats were divided into 4 series (A-D) of the experimental groups (n=7). In series A the importance of peripherally administered ghrelin at different time of I/R period was studied. In series B the importance of centrally administered ghrelin at different time of I/R period was evaluated. In series C and D, the mechanisms of peripherally and centrally administered hormone were examined, respectively. Two models of the I/R period were selected: short lasting (30/60 min) and long lasting (60/120 min). The following drugs were used: ghrelin (50 μg/kg i.p. or 1 nmol in 10 μl i.c.v.), 6 hydroxy dopamine (50 mg/kg i.p.), nadolol (0.5 mg/kg i.p.), calcitonin gene related peptide fragment (CGRP(8-37), 100 μg /kg i.p.), capsaicin (5-10 mg/100 ml solution s.c.). The mesenteric blood flow (MBF-ml/min), the intestinal microcirculatory blood flow (LDBF-PU), the arterio-venous oxygen difference (AVO(2)-ml/O(2)/100 ml blood), and the intestinal oxygen uptake (VO(2)) in ml O(2)/min were measured. Mucosal impairment was assessed planimetrically with the use of a digital photo analyzer (LA) and histologically with the use of the six-point Park/Chiu scale. Peripheral administration of ghrelin evoked marked increase of MBF and LDBF by 42% and 48%, respectively, with significant reduction of LA by 38%. When ghrelin was administered at the beginning of the reperfusion period during the short I/R period or prior to the long lasting I/R period, the vascular reactions and protective effects were reduced, but not completely abolished. The central administration of ghrelin before the short I/R period significantly increased the MBF and LDBF by about 32% and 35%, respectively, as well as LA reduction by about 20% in comparison to the control group. However, when ghrelin was administered prior to the long I/R period or after the onset of completed ischemia, neither vascular nor protective effects were noticed. Sensory denervation and the blockade of the CGRP1 receptors totally blocked the protective and hyperemic effects of the peripherally administered ghrelin. Selective blockade of the adrenergic system or blunting of the vagal nerves (vagotomy) significantly but not totally eliminated the effects of centrally applied ghrelin, which were abolished when both adrenergic and parasympathetic pathways were ablated. These results indicate that ghrelin applied centrally or peripherally markedly increases resistance of the intestinal tissue during the I/R period induced mucosal and hyperemic impairment evoked by I/R. Ghrelin is an important mediator of the increase in the intestinal microcirculation and elevation of the intestinal metabolism, which seems to be, at least in part, responsible for the observed protection of the intestine subjected to I/R. Impairment of this microvasculature response due to I/R seems to be responsible for a markedly observed weaker effect of ghrelin when this hormone was administered after the ischemic period. The lack of a protective effect observed after central administration of this peptide against a long lasting I/R period is probably due to damage of neural pathways caused by I/R. Finally, the peripheral activity of ghrelin in the intestine is mediated by the sensory neurons with a prominent role of CGRP released from their endings. However, this peripheral action of ghrelin depends upon the proper functioning of both the sympathetic and parasympathetic system.     

Atorvastatin Attenuates Ischemia/Reperfusion-Induced Hippocampal Neurons Injury Via Akt-nNOS-JNK Signaling Pathway

Ischemia-induced brain damage leads to apoptosis like delayed neuronal death in selectively vulnerable regions, which could further result in irreversible damages. Previous studies have demonstrated that neurons in the CA1 area of hippocampus are particularly sensitive to ischemic damage. Atorvastatin (ATV) has been reported to attenuate cognitive deficits after stroke, but precise mechanism for neuroprotection remains unknown. Therefore, the aims of this study were to investigate the neuroprotective mechanisms of ATV against ischemic brain injury induced by cerebral ischemia reperfusion. In this study, four-vessel occlusion model was established in rats with cerebral ischemia. Rats were divided into five groups: sham group, I/R group, I/R+ATV group, I/R+ATV+LY, and I/R+SP600125 group. Cresyl violet staining was carried out to examine the neuronal death of hippocampal CA1 region. Immunoblotting was used to detect the expression of the related proteins. Results showed that ATV significantly protected hippocampal CA1 pyramidal neurons against cerebral I/R. ATV could increase the phosphorylation of protein kinase B (Akt1) and nNOS, diminished the phosphorylation of JNK3 and c-Jun, and further inhibited the activation of caspase-3. Whereas, all of the aforementioned effects of ATV were reversed by LY294002 (an inhibitor of Akt1). Furthermore, pretreatment with SP600125 (an inhibitor of JNK) diminished the phosphorylation of JNK3 and c-Jun, and further inhibited the activation of caspase-3 after cerebral I/R. Taken together, our results implied that Akt-mediated phosphorylation of nNOS is involved in the neuroprotection of ATV against ischemic brain injury via suppressing JNK3 signaling pathway that provide a new experimental foundation for stroke therapy.     

Suppression of intestinal mucosal apoptosis by ghrelin in fasting rats

Ghrelin is mainly produced in the stomach and has several physiologic functions. The aim of this study was to investigate whether ghrelin regulates apoptosis in the small intestinal mucosa of fasting rats. Intestinal mucosal apoptosis was evaluated as the percentage of fragmented DNA, villus height, and terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end-labeling (TUNEL) staining and by Western blot analysis of caspase-3 in 48-hr fasting rats. Crypt cell proliferation was evaluated by counting the number of 5-bromo-2-deoxyuridine (BrdU) positive cells. Ghrelin was administered intraperitoneally at dosages of 2.5, 25, and 250 microg/kg per 48 hrs by continuous infusion via an Alzet micro-osmotic pump or injections at 12-hr intervals. Ghrelin was also infused in rats that underwent truncal vagotomy. The lowest dosage of ghrelin (2.5 microg/kg per 48 hrs) was administered into the third cerebroventricle. Ghrelin treatment attenuated the percentage of fragmented DNA in the small intestinal mucosa in 48-hr fasting rats in a dose-dependent manner. Continuous infusion of ghrelin and injections of ghrelin at 12-hr intervals suppressed intestinal apoptosis almost equally. This effect on apoptosis was not attenuated by truncal vagotomy. Cerebroventricular infusion of ghrelin also attenuated intestinal apoptosis. The antiapoptotic effect of ghrelin was confirmed by decreased TUNEL staining, recovery of the villus height, and decreased expression of caspase-3. BrdU uptake indicated that ghrelin enhanced cell proliferation in the intestinal crypt. Taken together, these data indicate that ghrelin enhanced intestinal growth with the suppression of small intestinal mucosal apoptosis in 48-hr fasting rats, suggesting that ghrelin controls intestinal function through the regulation of intestinal apoptosis.     

Ghrelin stimulates food intake and growth hormone release in rats with thermal injury: synthesis of ghrelin

Ghrelin, a 28-residue octanoylated peptide recently isolated from the stomach, exhibits anti-cachectic properties through regulating food intake, energy expenditure, adiposity, growth hormone secretion and immune response. Burn injury induces persistent hypermetabolism and muscle wasting. We therefore hypothesized that ghrelin may also play a role in the pathophysiology of burn-induced cachexia. Overall ghrelin expression in the stomach over 10 days after burn was significantly decreased (p = 0.0003). Total plasma ghrelin was reduced 1 day after burn. Thus, changes in ghrelin synthesis and release may contribute to burn-induced dysfunctions. Ghrelin (30 nmol/rat, i.p.) greatly stimulated 2 h food intake in rats on five separate days after burn and in control rats. On post-burn day 15, plasma growth hormone levels were significantly lower than in controls, and this was restored to normal levels by ghrelin (10 nmol/rat, i.p.). These observations suggest that ghrelin retains its ability to favorably modulate both the peripheral anabolic and the central orexigenic signals, even after thermal injury despite ongoing changes due to prolonged and profound hypermetabolism, suggesting that long-term treatment with ghrelin may attenuate burn-induced dysfunctions.     

肢体缺血预处理减少大鼠全脑缺血再灌注诱导的海马CA1区锥体神经元凋亡

探探讨肢体缺血预处理(limb ischemic preconditioning,LIP)对大鼠全脑缺血再灌注后海马CA1区锥体细胞凋亡的影响。46只大鼠椎动脉凝闭后分为假手术组、肢体缺血组、脑缺血组、LIP组。重复夹闭大鼠双侧股动脉3次(每次10min,间隔10min)作为LIP,之后立即夹闭双侧颈总动脉进行全脑缺血8min后再灌注。DNA凝胶电泳、TUNEL和吖啶橙/溴乙锭(AO/EB)双染技术从生化和形态学方面观察海马神经元凋亡的情况。凝胶电泳显示,脑缺血组出现了凋亡特征性DNA梯状条带,而LIP组无上述条带出现。与脑缺血组比较,LIP可明显减少海马CAI区TUNEL阳性神经元数(17.8±5.8vs 69.8±12,P<0.01)。AO/EB染色也显示LIP可明显减少脑缺血再灌注引起的神经元凋亡。以上结果提示,LIP可抑制脑缺血再灌注后海马神经元的凋亡,进而减轻脑缺血再灌注损伤,提供脑保护作用。     

Reduced ghrelin production induced anorexia after rat gastric ischemia and reperfusion

The gastrointestinal (GI) tract is one of the most susceptible organs to ischemia. We previously reported altered gastric motility after gastric ischemia and reperfusion (I/R). However, there have also been few reports of alterations in the eating behavior after gastric I/R. Ghrelin is a GI peptide that stimulates food intake and GI motility. Although ghrelin itself has been demonstrated to attenuate the mucosal injuries induced by gastric I/R, the endogenous ghrelin dynamics after I/R has not yet been elucidated. The present study was designed to investigate the relationship between food intake and the ghrelin dynamics after gastric I/R. Wistar rats were exposed to 80-min gastric ischemia, followed by 12-h or 48-h reperfusion. The food intake, plasma ghrelin levels, gastric preproghrelin mRNA expression levels, and the histological localization of ghrelin-immunoreactive cells were evaluated. The effect of exogenous ghrelin on the food intake after I/R was also examined. Food intake, the plasma ghrelin levels, the count of ghrelin-immunoreactive cells corrected by the percentage areas of the remaining mucosa, and the expression levels of preproghrelin mRNA in the stomach were significantly reduced at 12 h and 48 h after I/R compared with the levels in the sham-operated rats. Intraperitoneal administration of ghrelin significantly reversed the decrease of food intake after I/R. These data show that gastric I/R evoked anorexia with decreased plasma ghrelin levels and ghrelin production, which appears to be attributable to the I/R-induced gastric mucosal injuries. The decrease in the plasma ghrelin levels may have been responsible for the decreased food intake after gastric I/R.     

Butylphthalide Suppresses Neuronal Cells Apoptosis and Inhibits JNK–Caspase3 Signaling Pathway After Brain Ischemia /Reperfusion in Rats

Although Butylphthalide (BP) has protective effects that reduce ischemia-induced brain damage and neuronal cell death, little is known about the precise mechanisms occurring during cerebral ischemia/reperfusion (I/R). Therefore, the aim of this study was to investigate the neuroprotective mechanisms of BP against ischemic brain injury induced by cerebral I/R through inhibition of the c-Jun N-terminal kinase (JNK)–Caspase3 signaling pathway. BP in distilled non-genetically modified Soybean oil was administered intragastrically three times a day at a dosage of 15 mg/(kg day) beginning at 20 min after I/R in Sprague–Dawley rats. Immunohistochemical staining and Western blotting were performed to examine the expression of related proteins, and TUNEL-staining was used to detect the percentage of neuronal apoptosis in the hippocampal CA1 region. The results showed that BP could significantly protect neurons against cerebral I/R-induced damage. Furthermore, the expression of p-JNK, p-Bcl2, p–c-Jun, FasL, and cleaved-caspase3 was also decreased in the rats treated with BP. In summary, our results imply that BP could remarkably improve the survival of CA1 pyramidal neurons in I/R-induced brain injury and inhibit the JNK–Caspase3 signaling pathway.     

Human Ghrelin Mitigates Intestinal Injury and Mortality after Whole Body Irradiation in Rats

Widespread use of ionizing radiation has led to the realization of the danger associated with radiation exposure. Although studies in radiation countermeasures were initiated a half century ago, an effective therapy for a radiomitigator has not been identified. Ghrelin is a gastrointestinal hormone, and administration of ghrelin is protective in animal models of injuries including radiation combined injury. To test whether ghrelin can be protective in whole body irradiaton (WBI) alone, male Sprague Dawley (SD) rats were treated with human ghrelin (20 nmol/rat) daily for 6 days starting at either 24 h or 48 h after 10 Gray (Gy) WBI and survival outcome was examined. The 10 Gy WBI produced a LD^70/30 model in SD rats (30% survival in 30 days). The survival rate in rats treated with ghrelin starting at 24 h was significantly improved to 63% and when treatment was initiated at 48 h, the survival remained at 61%. At 7 days post WBI, plasma ghrelin was significantly reduced from the control value. Ghrelin treatment starting at 24 h after WBI daily for 6 days improved histological appearance of the intestine, reduced gut permeability, serum endotoxin levels and bacterial translocation to the liver by 38%, 42% and 61%, respectively at day 7 post WBI. Serum glucose and albumin were restored to near control levels with treatment. Ghrelin treatment also attenuated WBI-induced intestinal apoptosis by 62% as evidenced by TUNEL staining. The expression of anti-apoptotic cell regulator Bcl-xl was decreased by 38% in the vehicle and restored to 75% of the control with ghrelin treatment. Increased expression of intestinal CD73 and pAkt were observed with ghrelin treatment, indicating protection of the intestinal epithelium after WBI. These results indicate that human ghrelin attenuates intestinal injury and mortality after WBI. Thus, human ghrelin can be developed as a novel mitigator for radiation injury.     

姜黄素对自发性高血压大鼠脑缺血/再灌注后海马神经元损伤及RANTES表达的影响

目的：探讨姜黄素对自发性高血压大鼠（SHR）脑缺血/再灌注后认知功能及海马神经元损伤和调解活化正常T细胞表达和分泌的趋化因子（RANTES）表达的影响。方法：雄性Wistar-Kyoto大鼠（WKY）和SHR,随机分为5组：假手术组（W-Sham、S-Sham）、缺血/再灌注组（W-I/R、S-I/R）和姜黄素组（S-Cur）,各组按再灌注时间分为3h、12 h、1 d、3 d、7 d 5个亚组（n=6）。采用四血管阻断法制备全脑缺血/再灌注模型,HE染色观察海马CA1区神经细胞形态,Nissl染色计数海马CA1区平均锥体细胞密度,ELISA法检测海马RANTES表达,于再灌注后7 d观察行为学。结果：与假手术组大鼠比较,缺血/再灌注组大鼠学习和记忆能力下降,海马CA1区神经元损伤加重,海马RANTES蛋白表达上调（P〈0.05）;与W-I/R大鼠比较,S-I/R大鼠学习和记忆能力下降,海马CA1区神经元损伤加重,海马RANTES蛋白表达上调（P〈0.05）;姜黄素组大鼠学习和记忆能力明显改善,海马CA1区神经元损伤减轻,海马RANTES蛋白表达下调（P〈0.05）。结论：缺血/再灌注更易导致SHR海马神经元损伤。姜黄素减轻SHR脑缺血/再灌注海马神经元损伤,其机制可能与抑制RANTES蛋白的表达有关。     

Stimulation of neurogenesis in rat nucleus of the solitary tract by ghrelin

Ghrelin, a gastric hormone, regulates growth hormone secretion and energy homeostasis. The present study shows that ghrelin promotes neural proliferation in vivo and in vitro in the rat nucleus of the solitary tract (NTS). Systemic administration of ghrelin significantly increased 5-bromo-2'-deoxyuridine (BrdU) incorporation in the NTS in adult rats with cervical vagotomy. Cultured NTS neurons contain immature precursor cells as shown by expression of Hu protein. Exposure of cultured NTS neurons to ghrelin significantly increased the percentage of BrdU incorporation into cells in both dose- and time-dependent manners. Co-localization of Hu immunoreactivity with BrdU labeling was demonstrated by double fluorescent staining, suggesting that cells labeled with BrdU are neuronal cells. Ghrelin receptor mRNA was detected in tissues from the NTS. The mitotic effect of ghrelin was abolished by treatment of cultured NTS neurons with ghrelin receptor antagonists: D-Lys-3-GHRP-6 and [D-Arg1, D-Phe-5, D-Trp-7, 9, Leu-11] substance P. Diltiazem, a L-type calcium channel blocker, significantly attenuated ghrelin-mediated increments in BrdU incorporation. Ghrelin acts directly on NTS neurons to stimulate neurogenesis.     

Salvianolic acid A alleviated inflammatory response mediated by microglia through inhibiting the activation of TLR2/4 in acute cerebral ischemia-reperfusion

BackgroundToll-like receptor 2 and Toll-like receptor 4 (TLR2/4) on microglia have been found as important regulators in the inflammatory response during cerebral ischemia/reperfusion (I/R). In China, traditional Chinese medicine Salvia miltiorrhiza (danshen) and its some components are considered to be effective in rescuing cerebral I/R injury through clinical practice.Hypothesis/PurposeHere we examined the effect of Salvianolic acid A (SAA), a monomer compound in the water extract of Salvia miltiorrhiza, on TLR2/4 of microglia and its mediated inflammatory injury during cerebral I/R in vivo and in vitro.Study DesignFor exploring the effect of SAA on cerebral I/R and TLR2/4, classic middle cerebral artery occlusion (MCAO) model and oxygen glucose deprivation / reoxygenation (OGD/R) model of co-culture with primary hippocampal neurons and microglia in vitro were used. Signal pathway research and gene knockout have been applied to further explain its mechanism.MethodsThe evaluation indexes of I/R injury included infarct size, edema degree and pathology as well as primary hippocampal neurons and microglia culture, ELISA, western, RT-PCR, HE staining, immunofluorescence, flow cytometry, siRNA gene knockout were also employed.ResultsSAA significantly improved the degree of brain edema and ischemic area in I/R rats accompanied by decreases in levels of interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α). Pathological staining revealed that SAA could reduce inflammatory cell infiltration and mcirogila activation after reperfusion. Both protein and gene expression of TLR2 and TLR4 in ischemic hemisphere were obviously inhibited by SAA treatment while changes were not found in the non-ischemic hemisphere. In order to further study its mechanism, OGD/R model was used to mimic inflammatory damage of ischemic tissue by co-culturing primary rat hippocampal neurons and microglial cells. It was found that SAA also inhibited the protein and gene expression of TLR2 and TLR4 after OGD/R injury in microglia. After TLR2/4 knockout, the inhibitory effect of SAA on IL-1β and TNF-α levels in cell supernatant and neuron apoptosis were significantly weakened in each dose group. Moreover, expression levels of myeloid differentiation factor 88 (MyD88), NFκB, IL-1β and IL-6 in TLR2/4 mediated inflammatory pathway were reduced with SAA treatment.ConclusionSAA could significantly reduce the inflammatory response and injury in cerebral ischemia-reperfusion in vivo and in vitro, and its mechanism may be through the inhibition of TLR2/4 and its related signal pathway.     

大鼠脑缺血再灌注后神经元和星形胶质细胞凋亡的比较研究

目的比较研究大鼠局灶性脑缺血再灌注后神经元和星形胶质细胞的凋亡规律。方法建立大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)再灌注模型,在缺血再灌注后1、3、7、14d断头取脑,应用流式细胞分选技术和原位末端标记法分别检测各组MCAO后不同时期神经元和星形胶质细胞凋亡情况。结果局灶性脑缺血再灌注后,海马区星形胶质细胞凋亡数量超过神经元,其凋亡以再灌注3d最为显著,而神经元则以7d最为显著;而皮层区神经元凋亡数量超过星形胶质细胞,两种细胞凋亡均在再灌注后7d达高峰。结论脑缺血再灌注后,皮层和海马区的神经元及星形胶质细胞均可发生凋亡,海马区星形胶质细胞比皮层区更易凋亡,而皮层区神经元比海马区更易凋亡。