Fluoxetine regulates hippocampal synaptic plasticity in CUMS depression rats

AIM: To investigate the role of fluoxetine in the hippocampal synaptic plasticity in chronic unpredictable mild stress (CUMS) depression rats and its effect on mTOR and autophagy signaling pathways. METHODS: Male Sprague-Dawley rats (n=60) were randomly divided into normal control group, CUMS group and fluoxetine group. The CUMS rat model was established through CUMS combined with solitary raising, and fluoxetine (20 mg·kg^-1·d^-1) was administered via intragastric gavage. The changes of body weight, the ratio of sugar intake and the results of the behavioral test were recorded to identify the modeling. Moreover, the expression of synaptic plasticity-related proteins glial fibrillary acidic protein (GFAP) and synaptophysin (SYP), apoptosis-related proteins Bcl-2 and caspase-3, mTOR signaling proteins mTOR and 4EBP1, and autophagy-related proteins beclin 1 and LC3 were examined by RT-PCR and Western blot.RESULTS: Compared with control group, the body weight, sucrose intake, and total distance and intermediate residence time in the open field test were significantly decreased in CUMS group. The results of RT-PCR and Western blotting showed that the mRNA and protein levels of SYP and GFAP in CUMS group were significantly down-regulated compared with control group. The expression of Bcl-2 in CUMS group was downregulated, while the protein level of cleaved caspase-3 increased. Decreased phosphorylation levels of mTOR and its downstream target molecule 4EBP1 were observed in CUMS group. Besides, the autophagy-related proteins beclin 1 and LC3 were significantly upregulated at mRNA and protein levels. All these results(upregulation or downregulation) were attenuated by the treatment with fluoxetine, and the difference was statistically significant. CONCLUSION: Fluoxetine might improve hippocampal synaptic plasticity and alleviate symptoms of depression by supressing apoptosis/autophagy signaling pathways and upregulating mTOR signaling pathway.     

Effect of autophagy on necroptosis of renal tubular epithelial cells in subtotal nephrectomy rats

AIM: To explore whether autophagy is involved in the excessive death of renal tubular epithelial cells in subtotal nephrectomy(SNx) rats and the relationship between autophagy and necroptosis in the kidney of SNx rats. METHODS: Male Sprague-Dawley rats were randomly assigned to control group(n=6) and SNx group(n=42). The rats in SNx group were subjected to SNx. Sham surgery was performed in the rats in control group. The rats in SNx group were divided into subgroups at 0, 4, 8 and 12 weeks(n=6) and the other rats in SNx group were divided into SNx+vehicle group, SNx+necrostatin-1(Nec-1) group and SNx+3-methyladenine(3-MA) group. The expression of RIP1, RIP3, LC3 and beclin-1 at mRNA and protein levels was measured at 0, 4, 8 and 12 weeks by qPCR and immunohistochemistry. The effects of Nec-1 or 3-MA on the protein expression of LC3-I, LC3-II and beclin-1, and production of reactive oxygen species(ROS) in the rat kidney were determined by Western blot and DCFH-DA staining. The death of renal tubular epithelial cells in the SNx rats was observed by TUNEL staining and electron microscopy. Finally, the effects of Nec-1 and 3-MA on blood urea nitrogen(BUN), serum creatinine(SCr) and the pathological changes of the renal tissues were analyzed. RESULTS: The highest mRNA and protein levels of RIP1, RIP3, LC3 and beclin-1 appeared at the 8th week after SNx(P<0.01). Compared with the rats in SNx+vehicle group, the protein over-expression of LC3-II/I and beclin-1, renal tubular epithelial cells with typical morphological features of necroptotic cell death and TUNEL-positive renal tubular cells were decreased in the SNx rats treated with Nec-1 and 3-MA(P<0.01), but 3-MA did not reduce the increased concentration of ROS. In addition, treatment with Nec-1 and 3-MA obviously reduced BUN, SCr(P<0.05), glomerulosclerosis index and tubulointerstitial injury score(P<0.01). CONCLUSION: Autophagy participates in the excessive death of renal tubular epithelial cells in SNx rats. Inhibition of autograph prevents necroptotic cell death of renal tubular cells, and alleviates chronic renal injury in SNx rats.     

Autophagy induces mitochondrial dysfunction in neurons from neonatal rats with hypoxic ischemic encephalopathy

AIM: To explore the influence of autophagy on the induction of mitochondrial dysfunction in the neurons in a neonatal rat hypoxic ischemic encephalopathy (HIE) model. METHODS: Ten-day-old rat pups (n=30) were randomly divided into sham group and model group. The rats in the latter group were subject to hypoxia-ischemia treatment via unilateral common carotid artery ligation. The rats were sacrificed for brain pathological examination, and the protein levels of cleaved caspase-3 and LC3B-II were detected by immunohistochemical analysis. For the in vitro experiments, the autophagy of primarily cultured rat neurons was observed after hypoxia, and Western blot and mitochondrial function testing were also performed. RESULTS: Compare with sham group, the hypoxia-ischemia treatment caused atrophy and apoptosis of neurons, and ventricular area enlargement of rat brains. Immunohistochemical results demonstrated significantly higher levels of apoptosis- and autophagy-associated proteins, such as cleaved caspase-3 and LC3B-II (P<0.01). In vitro experiments demonstrated that hypoxia induced autophagy and apoptosis in the neurons. Compared with sham group, there were higher levels of reactive oxygen species and mitochondrial superoxide, and lower mitochondrial membrane potential in the model group (P<0.01). CONCLUSION: In neonatal HIE rat model, the hypoxia-induced mitochondrial dysfunction is related to apoptosis and autophagy. It will provide a new idea for administration of autopahgy inducer agents in treatment of HIE.     

Flavonoid-induced autophagy in hormone sensitive breast cancer cells

The activity of 8-prenylapigenin (8-PA) and its 3'-methoxylated analogue isocannflavin B (IsoB) was investigated in estrogen-dependent T47-D and estrogen-independent MDA-MB-231 human breast cancer cell lines. 8-PA showed a biphasic effect on T47-D cell proliferation, while no significant effect was observed on MDA-MB-231 cells. Conversely, IsoB exhibited only an inhibitory effect on T47-D cell proliferation, accompanied by the appearance of an intense intracytoplasmic vacuolization of autophagic origin. Moreover, biochemical analysis showed that IsoB reduced Akt phosphorylation and p21^Cip1 expression in T47-D cells. These data show that the prenylflavone moiety is a versatile platform for the induction and modulation of bioactivity.     

Starvation induces autophagy of hypertrophic scar fibroblasts

［ABSTRACT］AIM: To investigate the starvation-induced autophagy of hypertrophic scar fibroblasts. METHODS: Primary human fibroblasts from hypertrophic scars were isolated and the fibroblasts in logarithmic growth phase were cultured with amino acid-free Earle's balanced salt solution (EBSS) instead of the DMEM medium. The cells were collected at the time points of 0 h, 1 h, 2 h and 3 h after EBSS culture. The expression of microtubule-associated protein 1 light chain 3 (LC-3) and autophagy-related protein Beclin-1 was detected by Western blotting and qRT-PCR. Autopagosomes in fibroblasts were observed by electron microscopy and monodansylcadaverine (MDC) staining. RESULTS: The expression of LC3 and Beclin-1 increased at 1 h after starvation, reached to the highest level at 2 h after starvation, and then began to decline, which were still higher than that in control group. Compared with control group, the autopagosomes were observed in the fibroblasts under fluorescence microscope and election microscope at 2 h after starvation. CONCLUSION: Autophagy in fibroblasts of hypertrophic scars can be induced by starvation, which may be related to the formation of hypertrophic scars.     

Role of autophagolysosomal inhibitor NH4Cl in apoptosis of human cervical cancer HeLa cells induced by vitamin K3

AIM：To investigate the role of autophagolysosomal inhibitor ammonium chloride (NH4Cl) in the apoptosis of human cervical cancer HeLa cells induced by vitamin K3(VK3). METHODS：The HeLa cells were divided into 4 groups：control group, 30 μmol/L VK3 group, 10 mmol/L NH4Cl group and 30 μmol/L VK3+10 mmol/L NH4Cl group. The viability of HeLa cells in each group was measured by MTT assay. The changes of lysosomal membrane in HeLa cells were detected by acridine orange (AO) staining. Autophagic vacuoles in the cells were observed by staining with monodansylcadaverine (MDC). Intracellular acidification in HeLa cells was detected by BCECF-AM flow cytometry analysis. The cells were stained with Hoechst 33342 to determine the chromatin condensation and apoptotic rate. RESULTS：Compared with control group,no effect of NH4Cl on the viability of HeLa cells was observed. In VK3 group, the cell viability decreased. AO staining appeared no change, and the autophagic vacuoles and the cytoplasmic acidification were observed. Nuclear chromatin condensation and cell apoptosis in the cells were also detected. Compared with VK3 group, combination of NH4Cl and VK3 enhanced the lysosomal permeability, the formation of autophagic vacuoles and the cytoplasmic acidification, and exacerbated the apoptosis. CONCLUSION：Autophagolysosomal inhibitor NH4Cl exacerbates apoptosis in HeLa cells, indicating that autophagy exerts protective effect on the process of injury in HeLa cells induced by VK3.     

Histone deacetylase SIRT1 and cell autophagy

.Sirtuin 1 (SIRT1), one of the nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases, plays an important role in regulating cell cycle, cell aging, apoptosis and metabolism. Autophagy, a vital basic phenomenon that wildly exists in eukaryotic cells, plays an important part in waste scavenging, structure reestablishment, growth and development. In recent years, more and more attention has been paid to the connection between SIRT1 and autophagy. Clarifying the relationship between SIRT1 and autophagy will be of great importance in preventing and controlling aging-related diseases. This article overviews its research advancement.     

脊尾白虾翻译控制肿瘤蛋白基因TCTP克隆及自噬调控相关基因在卵巢发育期的表达

为深入了解脊尾白虾(Exopalaemon carinicauda)卵巢发育和卵黄蛋白原发生的分子机制,本研究克隆得到脊尾白虾翻译控制肿瘤蛋白基因TCTP,并结合自噬调控基因TCTP、Hif-1α、Beclin1和Bcl-2在卵巢发育期的表达水平,阐释脊尾白虾卵黄蛋白原合成过程中的分子调控特征。研究显示,脊尾白虾TCTP基因c DNA全长为732 bp,编码168个氨基酸,具有典型的TCTP1和TCTP2功能域以及PKC和TKⅡ等mTOR信号通路相关的磷酸化位点。同时发现,甲壳动物TCTP普遍缺乏在其他动植物中高度保守的C末端的cys残基。进化分析显示,脊尾白虾TCTP与中华绒螯蟹(Eriocheir sinensis)亲缘关系最近。4种自噬调控基因在脊尾白虾卵巢发育期的表达结果显示,肝胰腺TCTP基因从增殖期到产后恢复期呈递减趋势;肝胰腺Hif-1α、Beclin1和Bcl-2基因表达趋势相似,即从增殖期到小生长期高表达,大生长期极显著下降,表达量最低(P0.01),这与外源性卵黄蛋白原的合成趋势大致相反。这些自噬调控基因可能通过自噬作用共同调节外源性卵黄蛋白原的合成。卵巢TCTP基因在小生长期表达量最高;卵巢Hif-1α基因从增殖期到产后恢复期持续升高,这与内源性卵黄蛋白原表达趋势相似;卵巢Beclin1基因在大生长期表达量最高,与脊尾白虾卵巢Ec R表达趋势相似,与卵巢Bcl-2基因表达趋势相反,这些自噬调控基因可能通过自噬作用共同促进内源性卵黄蛋白的合成。本研究表明,自噬调控基因TCTP、Hif-1α、Beclin1和Bcl-2在脊尾白虾卵巢发育时期相互协调共同作用,可能通过自噬作用调节脊尾白虾卵黄蛋白原的合成和卵巢发育。     

Irbesartan alleviates hepatic steatosis in db/db mice by inducing auto-phagy

AIM: To investigate the effect of irbesartan on the fatty liver of db/db mice and whether autophagy is involved in the process. METHODS: Male db/db mice (n=24) were randomly divided into model group and irbesartan group, and 12 db/m mice with similar age and weight were selected as normal control group. After 16 weeks of intervention respectively, the fatty liver-related parameters including body weight, liver index, blood lipid, liver function and pathological changes in the liver were observed. The protein levels of p-PI3K, p-Akt, and p-mTOR, as well as Atg-7, beclin-1 and LC3B in the liver tissues were detected by Western blot, and the autophagosomes in the liver were observed under electron microscope. RESULTS: Compared with the model group, the body weight, liver index, blood lipids, alanine and aspartate aminotransferase were decreased in irbesartan group (P<0.05). Moreover, the pathological changes in the liver were significantly ameliorated in irbesartan group than that of model group. Importantly, the protein levels of p-PI3K, p-Akt and p-mTOR were decreased with irbesartan administration, while the expression of Atg-7, beclin-1 and LC3B-Ⅱ was increased(P<0.05), which resulted in a distinct increase in autophagosomes. CONCLUSION: Irbesartan alleviates hepatic steatosis in db/db mice by inhibiting the PI3K/Akt/mTOR signaling pathway and upregulating the protein expression of Atg-7, beclin-1 and LC3B-Ⅱ, thereby inducing autophagy in hepatocytes.     

灵芝三萜单体细胞保护与自噬诱导活性比较

比较不同灵芝三萜单体对PCI2细胞保护和自噬诱导活性的差异。结果表明,灵芝酸A、B、C、C2、DM、K对H2O2诱导的细胞损伤最大保护率分别为32．83％、12．63％、53．03％、30．30％、2．53％和25．25％;对Aβ25-35诱导的细胞损伤最大保护率分剐为6．74％、29．02％、23．81％、25．07％、10．15％和5．12％;自噬诱导最大阳性率分别达到22．33％、32．67％、35．50％、28．33％、11．83％和29．50％。不同灵芝三萜单体的活性也有明显的差异,且各单体在不同活性方面均未表现出一致的强弱关系。