Insulin-like growth factor-I-induced androgen receptor activation is mediated by the PI3K/Akt pathway in C2C12 skeletal muscle cells

Although insulin-like growth factor-I (IGF-I) and androgen receptor (AR) are well known effectors of skeletal muscle, the molecular mechanism by which signaling pathways integrating AR and IGF-I in skeletal muscle cells has not been previously examined. In this study, the role of PI3K/Akt on IGF-I-induced gene expression and activation of AR in skeletal muscle cells was investigated. C2C12 cells were treated with IGF-I in the absence or presence of inhibitors of PI3K/Akt pathway (LY294002 and Wortmannin). Inhibition of the PI3K/Akt pathway with LY294002 or Wortmannin led to a significant decrease in IGF-I-induced AR phosphorylation and total AR protein expression. Furthermore, IGF-I-induced AR mRNA and skeletal α-actin mRNA were blocked by LY294002 or Wortmannin. Confocal images showed that IGF-I-induced AR translocation from cytosol to nucleus was inhibited significantly in response to treatment with LY294002 or Wortmannin. The present results suggest that modulating effect of IGF-I on AR gene expression and activation in C2C12 mouse skeletal muscle cells is mediated at least in part by the PI3K/Akt pathway.     

Effects of increased accumulation of doxorubicin due to emodin on efflux transporter and LRP1 expression in lung adenocarcinoma and colorectal carcinoma cells

We investigated the eplerenone-induced, PI3K/Akt- and GSK-3β-mediated cardioprotection against ischemia/reperfusion (I/R) injury in diabetic rats. The study groups comprising diabetic rats were treated for 14 days with 150 mg/kg/day eplerenone orally and 1 mg/kg wortmannin (PI3K/Akt antagonist) intraperitoneally with eplerenone. On the 15th day, the rats were exposed to I/R injury by 20-min occlusion of the left anterior descending coronary artery followed by 30 min of reperfusion. The hearts were processed for biochemical, molecular, and histological investigations. The I/R injury in diabetic rats inflicted a significant rise in the oxidative stress and apoptosis along with a decrease in the arterial and ventricular function and the expressions of PI3K/Akt and GSK-3β proteins. Eplerenone pretreatment reduced the arterial pressure, cardiac inotropy, and lusitropy. It significantly reduced apoptosis and cardiac injury markers. The histology revealed cardioprotection in eplerenone-treated rats. Eplerenone up-regulated the PI3K/Akt and reduced the GSK-3β expression. The group receiving wortmannin with eplerenone was deprived eplerenone-induced cardioprotection. Our results reveal the eplerenone-induced cardioprotection against I/R injury in diabetic rats and substantiate the involvement of PI3K/Akt and GSK-3β pathways in its efficacy.     

IGF-I/PI3K/Akt and IGF-I/MAPK/ERK pathways in vivo in skeletal muscle are regulated by nutrition and contribute to somatic growth in the fine flounder

The insulin-like growth factor-I (IGF-I) is a key regulator of skeletal muscle growth in vertebrates, promoting mitogenic and anabolic effects through the activation of the MAPK/ERK and the PI3K/Akt signaling pathways. Nutrition also affects skeletal muscle growth, activating intracellular pathways and inducing protein synthesis and accretion. Thus, both hormonal and nutritional signaling regulate muscle mass. In this context, plasma IGF-I levels and the activation of both pathways in response to food were evaluated in the fine flounder using fasting and refeeding trials. The present study describes for the first time in a nonmammalian species that the MAPK/ERK and PI3K/Akt are activated by exogenous circulating IGF-I, as well as showing that the MAPK/ERK pathway activation is modulated by the nutritional status. Also, these results show that there is a time-dependent regulation of IGF-I plasma levels and its signaling pathways in muscle. Together, these results suggest that the nutritionally managed IGF-I could be regulating the activation of the MAPK/ERK and the PI3K/Akt signaling pathways differentially according to the nutritional status, triggering different effects in growth parameters and therefore contributing to somatic growth in fish. This study contributes to the understanding of the nutrient regulation of IGF-I and its signaling pathways in skeletal muscle growth in nonmammalian species, therefore providing insight concerning the events controlling somatic growth in vertebrates.     

Bromelain induces cardioprotection against ischemia-reperfusion injury through Akt/FOXO pathway in rat myocardium

Bromelain (Br), a proteolytic enzyme extracted from the stem of the pineapple, is known to possess anti-inflammatory activity and has been shown to reduce blood viscosity, prevent the aggregation of blood platelets, and improve ischemia-reperfusion (I/R) injury in a skeletal muscle model. We investigated the capacity of Br to limit myocardial injury in a global I/R model. Adult male Sprague-Dawley rats were divided into two groups: control (PBS) and Br at 10 mg/kg in PBS administered via intraperitoneal injection (twice/day) for 15 consecutive days. On day 16, the hearts were excised and subjected to 30 min of global ischemia followed by 2 h of reperfusion. Br treatment showed higher left ventricular functional recovery throughout reperfusion compared with the controls [maximum rate of rise in intraventricular pressure (dP/dt max), 2,225 vs. 1,578 mmHg/s at 2 h reperfusion]. Aortic flow was also found to be increased in Br treatment when compared with that in untreated rats (11 vs. 1 ml). Furthermore, Br treatment reduced both the infarct size (34% vs. 43%) and the degree of apoptosis (28% vs. 37%) compared with the control animals. Western blot analysis showed an increased phosphorylation of both Akt and FOXO3A in the treatment group compared with the control. These results demonstrated for the first time that Br triggers an Akt-dependent survival pathway in the heart, revealing a novel mechanism of cardioprotective action and a potential therapeutic target against I/R injury.     

PI3K/Akt信号通路在白藜芦醇抗大鼠缺血/再灌注性心律失常中的作用及机制

目的：探讨磷脂酰肌醇-3-激酶/蛋白激酶B（PI3K/Akt）信号通路在白藜芦醇抗缺血/再灌注性心律失常中的作用及机制。方法：48只健康雄性SD大鼠,取心电图正常者随机分为4组（n=10）：假手术（SC组）组、缺血/再灌注（I/R组）组、白藜芦醇处理（Res处理组）组、PI3K抑制剂LY294002（LY294002组）组。建立大鼠在体心肌缺血/再灌注模型,观察各组心律失常的发生情况及左室血流动力学变化,Western blot法测定心肌组织中蛋白激酶B（Akt）、磷酸化蛋白激酶B（p-Akt）、缝隙连接蛋白43（Cx43）蛋白表达水平,以RT-PCR法从转录水平检测Cx43的表达水平。结果：与I/R组相比,Res处理组心律失常的发生率（心律失常评分）明显降低、左室舒缩功能明显升高,同时心肌Akt、Cx43蛋白表达及Cx43mRNA水平也明显升高;使用PI3K抑制剂LY294002后,心肌Akt、Cx43蛋白表达及Cx43mRNA水平下降的同时心律失常的发生率明显升高、左室舒缩功能明显降低。结论：白藜芦醇的抗再灌注性心律失常作用可能是通过激活PI3K/Akt信号通路,改变Cx43活性及分布实现的。     

Reduced hexokinase II impairs muscle function 2 wk after ischemia-reperfusion through increased cell necrosis and fibrosis

We previously demonstrated that hexokinase (HK) II plays a key role in the pathophysiology of ischemia-reperfusion (I/R) injury of the heart (Smeele et al. Circ Res 108: 1165-1169, 2011; Wu et al. Circ Res 108: 60-69, 2011). However, it is unknown whether HKII also plays a key role in I/R injury and healing thereafter in skeletal muscle, and if so, through which mechanisms. We used male wild-type (WT) and heterozygous HKII knockout mice (HKII(+/-)) and performed in vivo unilateral skeletal muscle I/R, executed by 90 min hindlimb occlusion using orthodontic rubber bands followed by 1 h, 1 day, or 14 days reperfusion. The contralateral (CON) limb was used as internal control. No difference was observed in muscle glycogen turnover between genotypes at 1 h reperfusion. At 1 day reperfusion, the model resulted in 36% initial cell necrosis in WT gastrocnemius medialis (GM) muscle that was doubled (76% cell necrosis) in the HKII(+/-) mice. I/R-induced apoptosis (29%) was similar between genotypes. HKII reduction eliminated I/R-induced mitochondrial Bax translocation and oxidative stress at 1 day reperfusion. At 14 days recovery, the tetanic force deficit of the reperfused GM (relative to control GM) was 35% for WT, which was doubled (70%) in HKII(+/-) mice, mirroring the initial damage observed for these muscles. I/R increased muscle fatigue resistance equally in GM of both genotypes. The number of regenerating fibers in WT muscle (17%) was also approximately doubled in HKII(+/-) I/R muscle (44%), thus again mirroring the increased cell death in HKII(+/-) mice at day 1 and suggesting that HKII does not significantly affect muscle regeneration capacity. Reduced HKII was also associated with doubling of I/R-induced fibrosis. In conclusion, reduced muscle HKII protein content results in impaired muscle functionality during recovery from I/R. The impaired recovery seems to be mainly a result of a greater susceptibility of HKII(+/-) mice to the initial I/R-induced necrosis (not apoptosis), and not a HKII-related deficiency in muscle regeneration.     

Sulodexide attenuates endoplasmic reticulum stress induced by myocardial ischaemia/reperfusion by activating the PI3K/Akt pathway

Acute myocardial ischaemia/reperfusion (MI/R) injury causes severe arrhythmias with a high rate of lethality. Extensive research focus on endoplasmic reticulum (ER) stress and its dysfunction which leads to cardiac injury in MI/R Our study evaluated the effects of sulodexide (SDX) on MI/R by establishing MI/R mice models and in vitro oxidative stress models in H9C2 cells. We found that SDX decreases cardiac injury during ischaemia reperfusion and decreased myocardial apoptosis and infarct area, which was paralleled by increased superoxide dismutase and reduced malondialdehyde in mice plasm, increased Bcl‐2 expression, decreased BAX expression in a mouse model of MI/R. In vitro, SDX exerted a protective effect by the suppression of the ER stress which induced by tert‐butyl hydroperoxide (TBHP) treatment. Both of the in vivo and in vitro effects were involved in the phosphatidylinositol 3‐kinase (PI3K)/Akt signalling pathway. Inhibition of PI3K/Akt pathway by specific inhibitor, LY294002, partially reduced the protective effect of SDX. In short, our results suggested that the cardioprotective role of SDX was related to the suppression of ER stress in mice MI/R models and TBHP‐induced H9C2 cell injury which was through the PI3K/Akt signalling pathway.     

Schisantherin A protects renal tubular epithelial cells from hypoxia/reoxygenation injury through the activation of PI3K/Akt signaling pathway

Schisantherin A (SchA), a dibenzocyclooctadiene lignan isolated from the fruit of Schisandra sphenanthera, was reported to possess anti‐inflammatory and antioxidant activities. However, its protective effect against renal ischemia‐reperfusion (I/R) injury in human renal tubular epithelial cells subjected to hypoxia/reoxygenation (H/R) has never been studied. Thus, herein, we investigated the effect of SchA on renal I/R injury in vitro. Our results demonstrated that SchA pretreatment significantly improved HK‐2 cell viability exposed to H/R. Pretreatment with SchA markedly inhibited the levels of reactive oxygen species and malondialdehyde, as well as suppressed the production of tumor necrosis factor‐α (TNF‐α), interleukin‐1β, and interleukin‐6 in H/R‐stimulated HK‐2 cells. In addition, SchA also suppressed H/R‐induced HK‐2 cell apoptosis. Furthermore, this protective effect of SchA was mediated through the PI3K/Akt signaling pathway in HK‐2 cells. These findings showed that SchA may exert a protective effect on renal tubular epithelial cells against H/R injury through the activation of PI3K/Akt signaling pathway.     

Cardiomyocyte protective effects of thyroid hormone during hypoxia/reoxygenation injury through activating of IGF-1-mediated PI3K/Akt signalling

Ischaemia/reperfusion (I/R) injury is a common clinical condition that results in apoptosis and oxidative stress injury. Thyroid hormone was previously reported to elicit cardiac myocyte hypertrophy and promote cardiac function after cardiac injury. We used an in vivo mouse model of I/R injury and in vitro primary cardiomyocyte culture assays to investigate the effects of thyroid hormone on cardiomyocytes during hypoxia/reoxygenation (H/R) injury. The results showed that T3 pretreatment in vivo significantly improved left ventricular function after I/R injury. In vitro, T3 pretreatment decreased cell apoptosis rate, inhibited caspase-3 activity and decreased the Bax/Bcl-2 ration induced by H/R injury. T3 pretreatment significantly attenuated the loss of mitochondrial membrane potential. Furthermore, it was observed that T3 diminished the expression of NCX1 protein and decreased SERCA2a protein expression in H/R-induced cardiomyocytes, and T3 prevented intracellular Ca²⁺ increase during H/R injury. Also, T3 increased the expression of IGF-1, and PI3K/Akt signalling in cardiomyocytes under H/R-induced injury, and that the protective effect of T3 against H/R-induced injury was blocked by the PI3K inhibitor LY294002. IGF-1 receptor (IGF-1R) inhibitor GSK1904529A significantly inhibited the expression of IGF-1R and PI3K/Akt signalling. In summary, T3 pretreatment protects cardiomyocytes against H/R-induced injury by activating the IGF-1-mediated PI3K/Akt signalling pathway.     

Des-acyl-ghrelin (DAG) normalizes hyperlactacidemia and improves survival in a lethal rat model of burn trauma

Critical illness, including burn injury, results in elevated plasma lactate levels. Dysregulation of PI3K/Akt signaling has been shown to play a predominant role in the inactivation of skeletal muscle PDC and, hence, in hyperlactacidemia in rat models of sepsis and endotoxemia. This observation, and our previous finding that DAG can reverse burn-induced skeletal muscle proteolysis through the activation of PI3K/Akt pathway, led us to hypothesize that DAG may also attenuate hyperlactacidemia in burn injury. Our investigations revealed that burn injury significantly elevated both skeletal muscle lactate production and plasma lactate levels. Moreover, this was accompanied in skeletal muscle by a 5–7 fold increase in mRNA expression of pyruvate dehydrogenase kinases (PDK) 2 and 4, and a ∼30% reduction in PDC activity. DAG treatment of burn rats completely normalized not only the mRNA expression of the PDKs and PDC activity, but also hyperlactacidemia within 24 h of burn injury. DAG also normalized epinephrine-induced lactate production by isolated skeletal muscles from normal rats. Moreover, DAG also improved survival in a lethal rat model of burn trauma. These findings with DAG may have clinical implications because chances of survival for critically ill patients are greatly improved if plasma lactate levels are normalized within 24 h of injury.     

Hydroxysafflor Yellow A Protects Against Cerebral Ischemia–Reperfusion Injury by Anti-apoptotic Effect Through PI3K/Akt/GSK3β Pathway in Rat

Hydroxysafflor yellow A (HSYA) is the major active chemical component of the flower of the safflower plant, Carthamus tinctorius L. Previously, its neuroprotection against cerebral ischemia–reperfusion (I/R) injury was reported by anti-oxidant action and suppression of thrombin generation. Here, we investigate the role of HSYA in cerebral I/R-mediated apoptosis and possible signaling pathways. Male Wistar rats were subjected to transient middle cerebral artery occlusion for 2 h, followed by 24 h reperfusion. HSYA was administered via tail-vein injection just 15 min after occlusion. The number of apoptotic cells was measured by TUNEL assay, apoptosis-related proteins Bcl-2, Bax and the phosphorylation levels of Akt and GSK3β in ischemic penumbra were assayed by western blot. The results showed that administration of HSYA at the doses of 4 and 8 mg/kg significantly inhibited the apoptosis by decreasing the number of apoptotic cells and increasing the Bcl-2/Bax ratio in rats subjected to I/R injury. Simultaneously, HSYA treatment markedly increased the phosphorylations of Akt and GSK3β. Blockade of PI3K activity by wortmannin dramatically abolished its anti-apoptotic effect and lowered both Akt and GSK3β phosphorylation levels. Taken together, these results suggest that HSYA protects against cerebral I/R injury partly by reducing apoptosis via PI3K/Akt/GSK3β signaling pathway.     

PI3K/Akt 通路在电针预处理诱导脑缺血耐受中的机制研究

目的:通过观察电针预处理对磷脂酰肌醇3激酶/蛋白质丝氨酸苏氨酸激酶(PI3K/Akt)通路的变化以及该通路抑制剂对电针预处理的脑保护的影响,探讨电针预处理诱导脑缺血耐受的可能机制。方法:线栓法单侧阻断大脑中动脉120min,再灌注24h制备大鼠大脑局灶性缺血再灌注(I/R)模型;Western Blot检测Akt磷酸化水平的变化;侧脑室注射PI3K/Akt通路抑制剂LY294002;神经行为学评分(Garcia标准)及TTC染色检测脑梗死体积比评价脑损伤程度。结果:电针预处理使大鼠神经行为学评分增高,脑梗死体积比降低(P<0.05);可上调Akt磷酸化水平,I/R2h达高峰(P<0.05)。侧脑室注射PI3K/Akt抑制剂LY294002,拮抗电针预处理的脑保护作用(P<0.05)。结论:电针预处理增加Ak(tSer473)磷酸化水平,在缺血再灌注早期上调PI3K/Akt通路可能是诱导大鼠脑缺血耐受的产生的主要机制。     

Trpc1 ion channel modulates phosphatidylinositol 3-kinase/Akt pathway during myoblast differentiation and muscle regeneration

We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work, we used primary cell cultures and isolated muscles from Trpc1(-/-) and Trpc1(+/+) murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models, we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1(-/-) muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD, Myf5, and myogenin and of one of their targets, the developmental MHC (MHCd). Consequently, muscle tension was systematically lower in muscles from Trpc1(-/-) mice. Importantly, the PI3K/Akt/mTOR/p70S6K pathway, which plays a crucial role in muscle growth and regeneration, was down-regulated in regenerating Trpc1(-/-) muscles. Indeed, phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K, the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1(-/-) primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca(2+) or pretreated with EGTA-AM or wortmannin, suggesting that the entry of Ca(2+) through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo, and identify a Ca(2+)-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration.     

CpG-ODN,the TLR9 agonist,attenuates myocardial ischemia/reperfusion injury: Involving activation of PI3K/Akt signaling

BackgroundToll-like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. The TLR9 ligand, CpG-ODN has been reported to improve cell survival. We examined effect of CpG-ODN on myocardial I/R injury.MethodsMale C57BL/6 mice were treated with either CpG-ODN, control-ODN, or inhibitory CpG-ODN (iCpG-ODN) 1 h prior to myocardial ischemia (60 min) followed by reperfusion. Untreated mice served as I/R control (n = 10/each group). Infarct size was determined by TTC straining. Cardiac function was examined by echocardiography before and after myocardial I/R up to 14 days.ResultsCpG-ODN administration significantly decreased infarct size by 31.4% and improved cardiac function after myocardial I/R up to 14 days. Neither control-ODN nor iCpG-ODN altered I/R-induced myocardial infarction and cardiac dysfunction. CpG-ODN attenuated I/R-induced myocardial apoptosis and prevented I/R-induced decrease in Bcl2 and increase in Bax levels in the myocardium. CpG-ODN increased Akt and GSK-3β phosphorylation in the myocardium. In vitro data suggested that CpG-ODN treatment induced TLR9 tyrosine phosphorylation and promoted an association between TLR9 and the p85 subunit of PI3K. Importantly, PI3K/Akt inhibition and Akt kinase deficiency abolished CpG-ODN-induced cardioprotection.ConclusionCpG-ODN, the TLR9 ligand, induces protection against myocardial I/R injury. The mechanisms involve activation of the PI3K/Akt signaling pathway.     

bFGF attenuates endoplasmic reticulum stress and mitochondrial injury on myocardial ischaemia/reperfusion via activation of PI3K/Akt/ERK1/2 pathway

Extensive research focused on finding effective strategies to prevent or improve recovery from myocardial ischaemia/reperfusion (I/R) injury. Basic fibroblast growth factor (bFGF) has been shown to have therapeutic potential in some heart disorders, including ischaemic injury. In this study, we demonstrate that bFGF administration can inhibit the endoplasmic reticulum (ER) stress and mitochondrial dysfunction induced in the heart in a mouse model of I/R injury. In vitro, bFGF exerts a protective effect by inhibiting the ER stress response and mitochondrial dysfunction proteins that are induced by tert‐Butyl hydroperoxide (TBHP) treatment. Both of these in vivo and in vitro effects are related to the activation of two downstream signalling pathways, PI3K/Akt and ERK1/2. Inhibition of these PI3K/Akt and ERK1/2 pathways by specific inhibitors, LY294002 and PD98059, partially reduces the protective effect of bFGF. Taken together, our results indicate that the cardioprotective role of bFGF involves the suppression of ER stress and mitochondrial dysfunction in ischaemic oxidative damage models and oxidative stress‐induced H9C2 cell injury; furthermore, these effects underlie the activation of the PI3K/Akt and ERK1/2 signalling pathways.     

Apelin-13 protects the heart against ischemia-reperfusion injury through inhibition of ER-dependent apoptotic pathways in a time-dependent fashion

Endoplasmic reticulum (ER) stress is activated during and contributes to ischemia-reperfusion (I/R) injury. Attenuation of ER stress-induced apoptosis protects the heart against I/R injury. Using apelin, a ligand used to activate the apelin APJ receptor, which is known to be cardioprotective, this study was designed to investigate 1) the time course of changes in I/R injury after ER stress; 2) whether apelin infusion protects the heart against I/R injury via modulation of ER stress-dependent apoptosis signaling pathways; and 3) how phosphatidylinositol 3-kinase (PI3K)/Akt, endothelial nitric oxide synthase (eNOS), AMP-activated protein kinase (AMPK), and ERK activation are involved in the protection offered by apelin treatment. The results showed that, using an in vivo rat I/R model induced by 30 min of ischemia followed by reperfusion, infarct size (IS) increased from 2 h of reperfusion (34.85 ± 2.14%) to 12 h of reperfusion (48.98 ± 3.35, P < 0.05), which was associated with an abrupt increase in ER stress-dependent apoptosis activation, as evidenced by increased CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, and JNK activation (CHOP: 2.49-fold increase, caspase-12: 2.09-fold increase, and JNK: 3.38-fold increase, P < 0.05, respectively). Administration of apelin at 1 μg/kg not only completely abolished the activation of ER stress-induced apoptosis signaling pathways at 2 h of reperfusion but also significantly attenuated time-related changes at 24 h of reperfusion. Using pharmacological inhibition, we also demonstrated that PI3K/Akt, AMPK, and ERK activation were involved in the protection against I/R injury via inhibition of ER stress-dependent apoptosis activation. In contrast, although eNOS activation played a role in decreasing IS at 2 h of reperfusion, it failed to modify either IS or ER stress-induced apoptosis signaling pathways at 24 h after reperfusion.     

Cryptotanshinone inhibits hypoxia/reoxygenation-induced oxidative stress and apoptosis in renal tubular epithelial cells

Cryptotanshinone (CTS), an active component extracted from the root of Salvia miltiorrhiza Bunge , was reported to attenuate hepatic ischemia/reperfusion (I/R) injury. However, its protective effect against renal I/R injury remains unclear. In this study, the role of CTS in renal I/R injury in vitro and its possible mechanism were investigated. Our results showed that CTS improved cell viability in HK-2 cells exposed to hypoxia/reoxygenation (H/R). CTS also inhibited the H/R-mediated production of reactive oxygen species, as well as increased the activities of superoxide dismutase and catalase in H/R-stimulated HK-2 cells. In addition, CTS dramatically attenuated the induction of bax expression and caspase-3 activity and alleviated the reduction of bcl-2 expression in HK-2 cells cultured with H/R. Furthermore, CTS activated the levels of p-PI3K and p-Akt in H/R-injured HK-2 cells; meanwhile, the renal protective activity of CTS was inhibited by the inhibitor of the (phosphatidylinositol 3 kinase/protein kinase B) PI3K/Akt pathway (LY294002). These findings indicate that CTS can ameliorate renal I/R injury in vitro partly through regulating the PI3K/Akt pathway.     

Neuroprotective effect and underlying mechanism of sodium danshensu [3-(3,4-dihydroxyphenyl) lactic acid from Radix and Rhizoma Salviae miltiorrhizae = Danshen] against cerebral ischemia and reperfusion injury in rats

Sodium danshensu (SDSS), the sodium salt of danshensu (DSS), has the same pharmacological effects as DSS. In the present study, we aimed to investigate the neuroprotective effect and possible mechanism of SDSS against cerebral ischemic/reperfusion injury. Sprague-Dawley rats were randomly divided into four groups: sham, control, 30 mg/kg and 60 mg/kg SDSS. Cerebral ischemia was induced by 2 h of middle cerebral artery occlusion (MCAO). Neurological functional deficits were evaluated according to the modified neurological severity score (mNSS); cerebral infarct volume and histological damage were measured by TTC or H–E staining. In addition, the number of apoptotic cells and caspase 3/7 activity were assessed by TUNEL or Caspase-Glo assay. And the expression of apoptosis-regulatory proteins and the PI3K/Akt pathway were investigated by western blotting. Our results showed that treatment with SDSS for 5 days after MCAO remarkably improved neurologic deficits and survival rate, reduced infarct volume and the number of dead neurons. SDSS also decreased the number of apoptotic cells, regulated the expression of Bcl-2 and Bax, and increased the ratio of Bcl-2/Bax. Further study revealed that treatment with SDSS also increased the level of p-Akt and p-GSK-3β. Taken together, our results suggest that SDSS has the neuroprotective effect against cerebral I/R injury, and the potential mechanism might to inhibition of apoptosis through activating the PI3K/Akt signal pathway.     

Overexpression of HSPA12B protects against cerebral ischemia/reperfusion injury via a PI3K/Akt-dependent mechanism

Background and purpose: HSPA12B is a newly discovered member of the Hsp70 family proteins. This study investigated the effects of HSPA12B on focal cerebral ischemia/reperfusion (I/R) injury in mice. Methods: Transgenic mice overexpressing human HSPA12B (Tg) and wild-type littermates (WT) were subjected to 60 min of middle cerebral artery occlusion to induce ischemia and followed by reperfusion (I/R). Neurological deficits, infarct volumes and neuronal death were examined at 6 and 24 hrs after reperfusion. Blood–brain-barrier (BBB) integrity and activated cellular signaling were examined at 3 hrs after reperfusion. Results: After cerebral I/R, Tg mice exhibited improvement in neurological deficits and decrease in infarct volumes, when compared with WT I/R mice. BBB integrity was significantly preserved in Tg mice following cerebral I/R. Tg mice also showed significant decreases in cell injury and apoptosis in the ischemic hemispheres. We observed that overexpression of HSPA12B activated PI3K/Akt signaling and suppressed JNK and p38 activation following cerebral I/R. Importantly, pharmacological inhibition of PI3K/Akt signaling abrogated the protection against cerebral I/R injury in Tg mice. Conclusions: The results demonstrate that HSPA12B protects the brains from focal cerebral I/R injury. The protective effect of HSPA12B is mediated though a PI3K/Akt-dependent mechanism. Our results suggest that HSPA12B may have a therapeutic potential against ischemic stroke.     

Curcumin treatment prevents increased proteasome and apoptosome activities in rat skeletal muscle during reloading and improves subsequent recovery

Immobilization is characterized by activation of the ubiquitin (Ub)-proteasome-dependent proteolytic system (UPS) and of the mitochondrial apoptotic pathway. Increased oxidative stress and inflammatory response occur in immobilized skeletal muscles. Curcumin exhibits antioxidant and anti-inflammatory properties, blocked proteasome activation in intact animals, and may favor skeletal muscle regeneration. We therefore measured the effects of curcumin on immobilization-induced muscle atrophy and subsequent recovery. Rats were subjected to hindlimb immobilization for 8 days (I8) and allowed to recover for 10 days (R10). Fifty percent of the rats were injected daily with either curcumin or vehicle. Proteolytic and apoptotic pathways were studied in gastrocnemius muscles. Curcumin treatment prevented the enhanced proteasome chymotrypsin-like activity and the trend toward increased caspase-9-associated apoptosome activity at I8 in immobilized muscles. By contrast, the increase of these two activities was blunted by curcumin at R10. Curcumin did not reduce muscle atrophy at I8 but improved muscle recovery at R10 and the cross-sectional area of muscle fibers of immobilized muscles. Curcumin reduced the increased protein levels of Smac/DIABLO induced by immobilization and enhanced the elevation of X-linked inhibitory apoptotic protein levels at R10. Ub-conjugate levels and caspase-3 activity increased at I8 and were normalized at R10 without being affected by curcumin treatment. Altogether, the data show that curcumin treatment improved recovery during reloading. The effect of curcumin during the atrophic phase on proteasome activities may facilitate the initiation of muscle recovery after reloading. These data also suggest that this compound may favor the initial steps of muscle regeneration.