Protective effects of rapamycin induced autophagy on CLP septic mice

Sepsis is a life-threatening condition that may develop to multiple organ failure and septic shock. Autophagy is considered to play an important role in the regulation of inflammation. The present study aims to investigate the protective role of mTORC1 inhibitor, rapamycin, on septic death using cecal ligation and puncture (CLP) mice model. Here, results showed that pretreatment with rapamycin reduced the pyroptosis of peritoneal macrophages stimulated by cecal contents and the release of inflammatory factors such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α); In septic mice, rapamycin treatment decreased the activation of inflammasome in lung, and alleviated the pathological injuries in lung, liver and spleen tissues during acute stage of sepsis. Treatment of rapamycin rescued animals from septic death significantly. Our results indicated that activation of autophagy is a potential strategy to regulate the excessive inflammation in acute stage of sepsis.     

Effect of remote ischemic postconditioning on autophagy of hippocampal neural cells after cardiopulmonary resuscitation in rats

AIM: To observe the effect of remote ischemic post-conditioning (RIPostC) on autophagy of hippocampal neural cells after cardiopulmonary resuscitation (CPR) in rats. METHODS: Male SD rats (n=45) were randomly divided into sham operation group (sham group), cardiac arrest (CA)/CPR group and RIPostC group, with 15 rats in each group. A CPR model of asphyxiated CA was established by clamping the tracheal tube. Neurological deficit scoring (NDS) was performed at different time points after return of spontaneous circulation (ROSC). The rats were sacrificed 24 h after ROSC and hippocampal tissues were removed. Western blot was used to detect autophagy markers LC3-Ⅱ/LC3-I and beclin-1 in the hippocampal tissues. The apoptosis was detected by TUNEL method. The formation of LC3 particles was observed by immunofluorescence. The ultrastructural changes of autophagosomes and mitochondria were observed under transmission electron microscope. RESULTS: Compared with sham group, the NDS scores of CA/CPR group were decreased, the protein expression of LC3-Ⅱ/LC3-I and beclin-1 was increased (P<0.05), and the apoptosis of the neural cells was increased (P<0.05). Compared with CA/CPR group, the NDS scores in RIPostC group was increased, the protein expression of LC3-Ⅱ/LC3-I and beclin-1 was decreased (P<0.05), and the neural cell apoptosis was decreased (P<0.05). The number of LC3 particles was decreased, intracellular autophagosome number was reduced, and the mitochondrial structure damage was alleviated. CONCLUSION: Remote ischemic post-conditioning improves neurological function in rats after CPR, which may be related to inhibition of excessive autophagy in hippocampus.     

Yunpiheluo decoction alleviates diabetic kidney injury by regulating Sirt1/AMPK signaling pathway and activating autophagy

AIM: To observe the effect on Yunpiheluo decoction (YPHL) on renal injury in type 2 diabetic rats and to explore the mechanism from the perspective of Sirt1-AMPK-autophagy. METHODS: Male Zucker diabetic fatty (ZDF) rats (n=24) were randomly divided into model group, Sirt1 over-expression group and YPHL group, and fed with high-fat and high-sugar diet for 10 weeks. ZL rats were used as normal control and fed with normal diet for 10 weeks. After 10 weeks, urine and blood were collected for renal function detection. The rats were sacrificed and specimen was submitted. In addition, the mRNA expression of Sirt1 was analyzed by real-time PCR. The protein levels of Sirt1, AMPK, p-AMPK, LC3 and P62 in the renal tissues wene determined by Western blot. The renal pathological changes were observed under light microscope with HE and Masson staining. RESULTS: Compared with control group, fasting blood glucose (FBG), urinary protein (UP), urinary albumin (U-ALB) and serum creatinine (SCr) in model group were obviously increased (P<0.01). The mRNA expression of Sirt1 was decreased (P<0.05). The protein levels of SIRT1, AMPK, p-AMPK and LC3-II/-I were decreased (P<0.01), and P62 was increased (P<0.01). Glomerular focal fibrosis, focal renal tubular epithelial cell vacuolation, necrosis, shedding and atrophy, tubular type, and renal interstitial fibrosis were observed. Compared with model group, FBG was obviously decreased in Sirt1 over-expression group (P<0.01), but it showed no significant change in YPHL group (P>0.05). SCr and U-ALB were decreased (P<0.05), Sirt1 mRNA was increased (P<0.05), the protein levels of SIRT1, AMPK, p-AMPK and LC3-II/-I were increased (P<0.01), and P62 was decreased (P<0.01) in Sirt1 over-expression group and YPHL group. HE and Masson staining showed that the renal damage in Sir1 over-expression group and YPHL group was attenuated. CONCLUSION: Yunpiheluo decoction may protect the kidney by increasing the expression level of Sirt1, activating AMPK, and regulating autophagy.     

类猪圆环病毒 P1诱导 PK15细胞自噬的研究

旨在探讨类猪圆环病毒P1感染对PK15细胞自噬的影响。将同一批PK15细胞分成对照组和感染组，感染组在类猪圆环病毒P1感染PK15细胞后14，24，48 h分别收集细胞，对照组也在同样时间收获，用透射电镜观察自噬现象的发生。结果表明，与对照组相比，感染组PK15细胞内出现典型自噬形态学变化。揭示类猪圆环病毒P1能诱导PK15细胞发生自噬，但细胞发生自噬的相关信号传导通路还有待进一步研究。     

Honokiol induces autophagy by inhibiting mTOR signaling pathway in lung cancer A549 cells

AIM:To investigate whether honokiol induces the autophagy of human lung cancer A549 cells and to explore its mechanism. METHODS:The A549 cells were cultured in vitro, and treated with honokiol at different concentrations (0, 10, 20, 40, 60 and 80 μmol/L) for 48 h. MTT assay was performed to analyze the effect of honokiol on the viability of the A549 cells. The formation of autophagosome was observed by staining with acridine orange under fluorescence microscope. The protein levels of autophagy-associated protein LC3, mTOR and p-mTOR in the A549 cells treated with honokiol, or combined with autophagy inhibitor 3-methyladenine (3-MA) were determined by Western blot. RESULTS:Honokiol significantly inhibited the viability of A549 cells in a dose-dependent manner (P<0.05). The number of the intracellular acidic autophageic vacuoles with bright red fluorescence was significantly increased after honokiol treatment. The protein level of LC3-Ⅱ/LC3-I in the A549 cells was significantly enhanced after honokiol (40 μmol/L) treatment, and the ratio of LC3-Ⅱ/LC3-I was significantly decreased by treatment with 3-MA (P<0.05). Furthermore, treatment with honokiol (40 μmol/L) in the A549 cells for 48 h also resulted in significant down-regulation of phosphorylated form of mTOR (P<0.01), while the total protein level was not changed. CONCLUSION:Honokiol significantly inhibits the growth of lung cancer A549 cells and induces the autophagy, which may be correlated with inhibition of mTOR signaling pathway.     

NOD8 inhibits autophagy in human pancreatic cancer cells and effect of apoptosis on autophagy regulatd by NOD8

AIM: To explore whether NOD8 inhibits autophagy in human pancreatic cancer cells and its underlying mechanisms, and to investigate the effect of apoptosis on the autophagy regulated by NOD8. METHODS: The empty plasmid pEGFP-C2 and recombinant plasmid pEGFP-NOD8 were transfected into the Panc-1 cells using JetPRIME reagent.The untransfected cells served as control group. The protein levels of NOD8, autophagy-related proteins beclin-1 and LC3-II, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway-related proteins Akt, p-Akt, mTOR and p-mTOR were determined by Western blot 48 h after transfection. Meanwhile, the number of LC3 spots was quantified by immunofluorescence staining. Furthermore, after a broad caspase inhibitor Z-VAD-FMK was applied to NOD8-over-expressing cells, the protein expression levels of beclin-1 and LC3-II were detected by Western blot and the number of LC3 spots was observed by immunofluorescence staining. RESULTS: The protein level of NOD8 in pEGFP-NOD8 group was significantly higher than that in control group and pEGFP-C2 group (P<0.01). The protein expression of beclin-1 and LC3-II, and the number of LC3 spots in pEGFP-NOD8 group were significantly decreased as compared with control group and pEGFP-C2 group. Moreover, the protein levels of p-AKT and p-mTOR in pEGFP-NOD8 group were higher than those in control group and pEGFP-C2 group, while no significant difference of mTOR and AKT protein expression was found among these 3 groups. Furthermore, the protein levels of beclin-1 and LC3-II, and the number of LC3 spots in pEGFP-NOD8+Z-VAD-FMK group were significantly increased compared with pEGFP-NOD8 group. CONCLUSION: NOD8 inhibits autophagy in the Panc-1 cells and its mechanism may be related to the activation of PI3K/Akt/mTOR pathways. Apoptosis enhances the inhibitory effect of NOD8 on autophagy.     

Mitophagy and heart failure

Heart failure (HF) is the end stage of various cardiovascular diseases. The prognosis of patients with HF is poor. The precise pathophysiological mechanism of HF is still not fully elucidated. Recent studies have shown that mitochondrial dysfunction is closely associated with HF. Mitophagy plays a crucial role in mitochondrial quality control. In this article, we review the present evidence implicating the role of mitophagy in HF, and provide the potential ther?apeutic strategies targeting mitophagy to improve outcome in the patients with HF.     

Destruction of autophagy marker LC3 rhythm is involved in β1-AA-induced H9c2 rat cardiomyocyte death

AIM To investigate the effect of β₁-adrenergic receptor autoantibodies （β₁-AA） on the rhythm of autophagy marker microtubule-associated protein 1 light chain 3 （LC3）, and the underlying mechanism of cardiomyocyte death. METHODS The test materials were Sprague-Dawley （SD） rats and H9c2 rat cardiomyocytes. The SD rats were randomly divided into immunization group and control group with 6 rats in each group. The H9c2 cells were randomly divided into control group, β₁-AA group, lentivirus （LV）-NC group, and LV-shPer2 group （n=6）. Affinity chromatography was used for purification of β₁-AA from rat serum. CCK-8 assay was used to observe the viability of cardiomyocytes treated with β₁-AA for 24 h. The cells were synchronized by dexamethasone and then treated with β₁-AA. The mRNA and protein levels of LC3 at different time points were determined by real-time PCR and Western blot, respectively. The Per2 protein level at different time points was also determined by by Western blot. JTK_CYCLE algorithm was used to estimate the circadian rhythm parameters. After destruction of LC3 circadian rhythm via LV-shPer2, CCK-8 assay was used to measure the viability of H9c2 cells. RESULTS High level of β₁-AA in rat serum was found after active immunization compared with control group （P<0.05）. The viability of H9c2 cells in β₁-AA group was significantly lower than that in control group （P<0.05）. The LC3 and Per2 rhythms were both disrupted in H9c2 cells induced by β₁-AA （JTK_CYCLE P<0.05）. After LV-shPer2 infection, the LC3 rhythm was disrupted （JTK_CYCLE P<0.05） and the cell viability was reduced （P<0.05）. CONCLUSION β₁-AA may induce the destruction of autophagy marker LC3 rhythm in rat cardiomyocytes and then promote cell death.