NLRP3 inflammasome in pathogenesis of MS/EAE and its targeted therapy

Inflammasomes, the important component of innate immune system, play an important role in inflammatory diseases. NLRP3, the most studied inflammasome, is activated after recognizing danger-associated molecular patterns and pathogen-associated molecular patterns. The activated NLRP3 inflammasome promotes inflammation by maturation and release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Involvement of the NLRP3 inflammasome in the development of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) was suggested in a number of studies. Therefore, targeting on NLRP3 inflammasome is one of the promising methods for treatment of related diseases. In this review, we summarize the main ways by which the NLRP3 inflammasome is activated in the cytosol. We also discuss the development and treatment of NLRP3 inflammasome in MS and EAE, and expect to provide reference for the treatment of MS.     

rhPGRN promotes cell proliferation by regulating autophagy and endoplasmic reticulum stress through MAPK/PI3K pathway

AIM To extract and purify recombinant human progranulin (rhPGRN) and to examine its effect on the proliferation, autophagy and endoplasmic reticulum stress (ERS) in human chondrocyte C28I2 and mouse macrophage RAW264.7. METHODS The histidine-tagged protein was specifically affinity-purified using Ni-NTA Sefinose resin, and the concentration and purity of the target protein were verified by Coomassie blue staining, BCA method and Western blot. The effects of rhPGRN on the proliferation, autophagy and ERS in C28I2 and RAW264.7 cells were detected by cell counting, Western blot and RT-qPCR. RESULTS The highly purified biologically active human recombinant protein rhPGRN was successfully extracted from the cell line with stable PGRN transfection. rhPGRN promoted the proliferation of the C28I2 cells and RAW264.7 cells, up-regulated the mRNA expression of cell cycle-related molecules (PCNA, cyclin B1 and cyclin D1) and the protein expression of Ki67, and increased the phosphorylation levels of proliferation-related signaling molecules ERK and Akt. Treatment with ERK pathway inhibitor U0126 inhibited rhPGRN-promoted proliferation, autophagy and ERS in the cells. The rhPGRN-induced autophagy of the cells was also inhibited by PI3K/Akt pathway inhibitor 3-methyladenine. The rhPGRN-promoted protein expression of Ki67 was down-regulated by autophagy inhibitor bafilomycin A1 and ERS inhibitor 4-phenylbutyric acid. CONCLUSION These results not only established a method for stable extraction of biologically active high-concentration high-purity recombinant protein rhPGRN, but also confirmed that the biological effect of rhPGRN on promoting cell proliferation was achieved through regulating autophagy and ERS via MAPK and PI3K/Akt pathway.     

Atorvastatin inhibits IL-1β and IL-18 releases via lowering the expression of NLRP1 inflammasome

AIM: To explore the role of nucleotide-binding oligomerization domain-like receptor protein 1 (NLRP1) inflammasome in atorvastatin-induced reduction of interleukin-1β (IL-1β) and interleukin-18 (IL-18) releases from the THP-1 macrophages. METHODS: Lipopolysaccharide (LPS, 10 μg/L) was used to trigger the secretion of IL-1β and IL-18 in the THP-1 macrophages. The cells were incubated with different concentrations of atorvastatin (1, 10 and 20 μmol/L) for 24 h, or treated with 10 μmol/L atorvastatin for different time (12 h, 24 h and 48 h). NLRP1 siRNA was transfected into the THP-1 cells. The mRNA expression of NLRP1 inflammasome was detected by RT-PCR. The protein expression of NLRP1 inflammasome was determined by Western blot. The secretion of proinflammatory cytokines IL-1β and IL-18 was quantified by ELISA. RESULTS: Atorvastatin inhibited the mRNA and protein expression of NLRP1 inflammasome in the THP-1 macrophages in a dose- and time-dependent manner. Transfection of NLRP1 siRNA significantly decreased the protein expression of NLRP1 and promoted the suppressive effect of atorvastatin on IL-1β and IL-18 secretion in the THP-1 macrophages. CONCLUSION: Atorvastatin inhibits the production of IL-1β and IL-18 in the macrophages through decreasing NLRP1 inflammasome expression, possibly contributing to the anti-inflammatory effect of atorvastatin on atherosclerosis.     

Effect of exogenous hydrogen sulfide on expression of NLRP3 inflammasome in hepatocytes

AIM: To evaluate the effect of exogenous hydrogen sulfide (H₂S) on the expression of NLRP3 inflammasome in hepatocytes.METHODS: The hepatocytes L02 and SMMC-7721 were used to establish the model of inflammation by stimulating with lipopolysaccharide (LPS) at different concentrations in vitro. The expression of NLRP3 inflammasome in the hepatocytes was detected by Western blot and the cell viability was measured by MTT assay for determining appropriate concentration of LPS. The hepatocytes were divided into 4 groups:the cells in control group were incubated with normal medium for 18.5 h; the cells in LPS group were incubated with normal medium for 0.5 h followed by 100 μg/L LPS for 18 h; the cells in LPS+H₂S group and H₂S group were incubated with 200 μmol/L sodium hydrosulfide hydrate (NaHS) for 0.5 h followed by 100 μg/L LPS or normal medium for 18 h, respectively. The protein expression of NLRP3 and caspase-1 in the cells of every group was determined by Western blot. RESULTS: Compared with control group, the protein expression of NLRP3 and caspase-1 increased significantly in LPS group (P<0.05) and had no significant change in H₂S group. Compared with LPS group, the protein expression of NLRP3 and caspase-1 in LPS+H₂S group decreased significantly (P<0.05). CONCLUSION: In hepatocytes, exogenous H₂S suppresses the expression of NLRP3 inflammasome.     

Glyburide reduces PFOS-induced lung injury in young rats through inhibiting NLRP3 inflammasome

AIM: To explore the possible mechanism of NLR family Pyrin domain-containing protein 3 (NLRP3) inflammasome involved in perfluorooctane sulfonate (PFOS)-induced lung injury in young rats. METHODS: Twenty-eight SD rats (21-day-old) were randomly divided into control (C) group, PFOS (P) group, glyburide (G) group and glyburide + PFOS (GP) group. PFOS exposure model and glyburide protection model were established. The lung specimens were collected for HE staining. The levels of myeloperoxidase (MPO) in the lung tissues, interleukin-1β (IL-1β) and interleukin-18 (IL-18) in the bronchoalveolar lavage fluid (BALF) were measured by ELISA. The concentration of PFOS in serum was measured by high-performance liquid chromatography (HPLC). The protein expression of NLRP3, caspase-1 and apoptosis-associated speck-like protein containing CARD (ASC) in the lung tissues was determined by Wes-tern blot. RESULTS: HE staining of lung tissues showed that compared with the control rats, there were obvious inflammatory infiltration in trachea and alveolar interstitium of the rats in P group. Glyburide reduced the inflammatory responses significantly. ELISA results showed that the level of MPO in the lung tissues of the rats in P group was higher than those in other 3 groups (P<0.05). The levels of IL-1β and IL-18 in the BALF of the rats in P group were significantly higher than those in control group and GP group (P<0.05). The results of Western blot showed that the protein levels of NLRP3, caspase-1 and ASC in P group were significantly higher than those in control group and GP group (P<0.01). Immunohistochemical staining results showed that compared with the other 3 groups, the expression of NLRP3 in P group was significantly increased (P<0.01). CONCLUSION: PFOS exposure may lead to lung injury in rats by activating NLRP3 inflammasome and then triggering inflammation, releasing inflammatory factors such as IL-1β. Glyburide specifically inhibits the assembly of NLRP3 inflammasome, suppresses the inflammatory responses and reduces the toxicity of PFOS in lung.     

Sulodexide protects against hyperglycemia-induced retinal vascular injury via suppressing NOX4/NLRP3 pathway

AIM To investigate the effect of sulodexide (SDX) on high glucose-induced damage in retinal microvascular endothelial cells. METHODS (1) High-fat diet combined with intraperitoneal injection of streptozocin were used to induce type 2 diabetes mellitus (DM) followed by injection of saline or SDX in C57BL/6J male mice. Retinal microvascular leakage and density, and the protein levels of NLRP3 inflammasome-related proteins, zonula occludens-1 (ZO-1) and NADPH oxidase 4 (NOX4) were measured. (2) Human retinal microvascular endothelial cells (HRMECs) were treated with normal glucose or high glucose with or without SDX, and were further transfected with siRNA to knock down NOX4, or infected by adenovirus to over-express NOX4. The protein levels of ZO-1, VE-cadherin (VE-Cad), NOX4 and NLRP3 inflammasome-related proteins as well as the level of reactive oxygen species (ROS) were detected. RESULTS Treatment with SDX increased the protein level of ZO-1, attenuated retinal leakage and NLRP3 inflammasome activation, and enhanced the density of microvasculature and the number of ganglion cells in diabetic retinas. The protein levels of ZO-1 and VE-Cad were decreased, while the levels of NOX4, NLRP3 inflammasome-related proteins and ROS generation were increased in high glucose-treated HRMECs. Silencing of NOX4 inhibited high glucose-induced increases in NLRP3 inflammasome and ROS generation, and decreases in the protein levels of ZO-1 and VE-Cad. Over-expression of NOX4 significantly increased the levels of NLRP3 inflammasome-related proteins and ROS generation in HRMECs, and reduced the protein levels of ZO-1 and VE-Cad. Treatment with SDX partly reversed NOX4 over-expression-induced changes. CONCLUSION SDX alleviates hyperglycemia-induced retinal microvascular endothelial injury via inhibiting NOX4/ROS/NLRP3 pathways.     

Involvement of TLR4/NLRP3 inflammasome in contrast medium-induced inflammation and injury in renal tubular epithelial cells

AIM: To investigate whether Toll-like receptor 4 (TLR4) and Nod-like receptor protein 3 (NLRP3) inflammasome were involved in contrast medium (CM)-induced inflammation and injury in renal tubular epithelial cells. METHODS: Iopromide was used to injure NRK-52E cells in the study. The cell viability was measured by CCK-8 assay. The protein levels of TLR4, NLRP3, apoptosis-associated speckle-like protein (ASC), caspase-1 and cleaved caspase-3 were determined by Western blot. The releases of interleukin (IL)-1β and IL-18 were detected by ELISA. The apoptotic rate was evaluated by Hoechst staining, and mitochondrial membrane potential (MMP) was analyzed by JC-1 staining. siRNA was transfected into the NRK-52E cells to silence NLRP3 expression. RESULTS: CM decreased the viability of NRK-52E cells (P<0.05). CM also elevated the protein levels of cleaved caspase-3, TLR4, NLRP3, IL-1β and IL-18 (P<0.05). Silencing NLRP3 attenuated CM-induced releases of inflammatory cytokines. Moreover, treatment with TLR4 inhibitor TAK-242 or knockdown of NLRP3 by siRNA transfection both attenuated cell apoptosis and loss of MMP caused by CM. CONCLUSION: TLR4/NLRP3 inflammasome takes part in the pathogenesis of CM-induced acute kidney injury, and mediates CM-induced injury and inflammation in renal tubular epithelial cells.     

Relationship between NLRP3 inflammasome-IL-1β axis and End-MT after acute myocardial infarction in rats

AIM: To investigate whether activation of NLRP3 inflammasome-IL-1β axis is consistent with endothelial-mesenchymal transition (End-MT) during the process of myocardial fibrosis after acute myocardial infarction (AMI). METHODS: Adult male SD rats (n=30) were randomly divided into sham operation group (n=15) and AMI group (n=15). After 28 d, Masson staining was used to detect the level of myocardial fibrosis. The activation of NLRP3 inflammasome including NLRP3, ASC, pro-caspase-1 and caspase-1, the endothelial cell markers CD31 and VE-cadherin, and the mesenchymal cell markers α-SMA and FSP1 were analyzed by Western blot. The expression of IL-1β was measured by ELISA. RESULTS: The levels of myocardial fibrosis and End-MT, the activation of NLRP3 inflammasome, and the expression of caspase-1 and IL-1β were significantly increased in AMI group compared with sham operation group (P<0.05). CONCLUSION: The activation of NLRP3 inflammasome-IL-1β axis is significantly consistent with End-MT process, suggesting that NLRP3 inflammasome-IL-1β, as a potential target for the activation of End-MT, will provide a novel theoretical target for the treatment of myocardial fibrosis and heart failure after AMI.     

Mycoplasma pneumoniae induces IL-1β production through activating NLRP3 inflammasome by ROS in RAW264.7 cells

AIM: To investigate whether Mycoplasma pneumoniae (Mp)-induced interleukin-1β (IL-1β) production in RAW264.7 cells is through the activation of NLRP3 inflammasome via reactive oxygen species (ROS). ME-THODS: RAW264.7 cells were randomly divided into 3 groups. In normal group, RAW264.7 cells were treated without Mp. In model group, RAW264.7 cells were treated with 1∶ 10 multiplicity of infection (MOI) of Mp. In NAC group, RAW264.7 cells were pretreated with N- acetylcysteine (NAC) at a concentration of 5 mmol/L for 30 min before infection with Mp. The RAW264.7cells were infected with Mp (1∶ 10 MOI) for 4, 8, 16 and 24 h in model group and NAC group, respectively. The intracellular ROS level was analyzed by flow cytometry. The mRNA expressions of NLRP3, ASC and caspase-1 were detected by real-time PCR. The protein levels of NLRP3, ASC and caspase-1 p20 were determined by Western blot. The levels of pro-inflammatory cytokine IL-1β in the supernatant were measured by ELISA. RESULTS: Compared with normal group, the production of ROS were significantly increased at 4, 8, 16 and 24 h after infection, the mRNA expression of NLRP3, ASC and caspase-1 were increased at 8, 16 and 24 h after infection, the protein levels of NLRP3, ASC and caspase-1 p20 were increased at 16 and 24 h after infection, and the releases of IL-1β were increased at 24 h after infection in model group (P<0.01). Compared with the model group, the level of ROS in NAC group decreased, so as the expression of NLRP3, ASC and caspase-1 at mRNA and protein levels and the releases of IL-1β in the supernatant at the corresponding time points. CONCLUSION: Mp may stimulate the ROS production to activate NLRP3 inflammasome in RAW264.7 cells.     

Activity of NLRP3 inflammasome in MRSA pneumonia secondary to influenza A virus infection in mice

AIM To evaluate the activity of NLRP3 inflammasome in methicillin-resistant Staphylococcus aureus （MRSA） pneumonia secondary to influenza A virus （IAV） HIN1 in mice. METHODS Pneumonia model caused by intranasal inoculation with only MRSA for 24 h （MRSA group） and with MRSA for 24 h secondary to IAV H1N1 infection for 6 d in advance （H1N1+MRSA group）in C57BL/6 mice were established.The mRNA expression of NLRP3, caspase-1 and interleukin-1β （IL-1β） in lung tissues was detected by RT-qPCR. The protein levels of NLRP3 and caspase-1 in the lung tissues were determined by Western blot. The serum concentration of IL-1β was measured by ELISA. The pathological changes of the lung tissues were examined. The correlation between rate of weight loss during infection and serum concentration of IL-1β was investigated. RESULTS In MRSA group, the mRNA levels and relative protein expression levels of NLRP3 and caspase-1 showed no difference compared with control group （P>0.05）, while the mRNA expression of IL-1β and the serum concentration of IL-1β were significantly higher than those in control group （P<0.01）. In H1N1+MRSA group, the mRNA levels and relative protein expression levels of NLRP3 and caspase-1 were significantly higher than those in control group, as well as higher than those in MRSA group （P<0.01）, the mRNA level and serum concentration of IL-1β were significantly higher than those in control group but lower than those in MRSA group （P<0.01）. The pathological observation of the lung in MRSA group showed inflammatory responses, and severer pneumonia in H1N1+MRSA group was found. The rate of weight loss in the mice of MRSA group and H1N1+MRSA group was negatively correlated with the serum concentration of IL-1β. CONCLUSION IL-1β expression induced by MRSA infection is in a NLRP3 inflammasome independent manner. It also suggests that IAV H1N1 infection in advance down regulates the expression of IL-1β in secondary infection with MRSA, which may contribute to the mechanism of MRSA pneumonia secondary to IAV infection.     

SO2 derivatives stimulate inflammation of airway epithelial cells through NLRP3 inflammasome

AIM:To investigate the effect of sulfur dioxide (SO₂) derivatives (sodium sulfite and sodium bisulfate) on NLRP3 inflammasome in airway epithelial cells. METHODS:SO₂ derivatives at different concentrations were applied to bronchial epithelial 16HBE cells for 12 h. The production of reactive oxygen species (ROS) was detected by flow cytometry. The protein levels of NLRP3 and caspase-1 p20 were analyzed by Western blot. The level of interleukin-1β(IL-1β) in the cell culture supernatant was measured by ELISA. The cell viability was measued by MTT assay, and the concentration of SO₂ derivatives used in the following experiments was 2 mmol/L. When the NLRP3 gene in 16HBE cells was silenced by RNA interference technique or N-acetyl cysteine (NAC) was used to pretreat 16HBE cells, the intracellular ROS was detected by flow cytometry, and the protein levels of NLRP3 and caspase-1 p20 and the secretion of IL-1β were determined by Western blot and ELISA, respectively. RESULTS:Compared with the control group, the level of intracellular ROS, the protein levels of NLRP3 and caspase-1 p20, and the secretion of IL-1β in cell supernatant were increased significantly in 2 mmol/L and 4 mmol/L SO₂ derivative groups (P<0.05). Compared with the 2 mmol/L group, the protein levels of NLRP3 and caspase-1 p20 were significantly inhibited in NLRP3 siRNA group (P<0.05). The concentration of IL-1β in the cell culture supernatant was significantly decreased (P<0.05). No significant difference of ROS level was observed. Significantly decreased protein levels of NLRP3 and caspase-1 p20, and the concentration of IL-1β in NAC group were found (P<0.05). CONCLUSION:SO₂ derivatives directly promote the production of IL-1β through NLRP3 inflammasome in bronchial epithelial cells.     

Secoisolariciresinol diglucoside attenuates renal inflammatory injury of mice induced by chronic intermittent hypoxia via inhibiting TXNIP and activating NLRP3 inflammasome

AIM To investigate the effectof flax lignan/secoisolariciresinol diglucoside （SDG） on the inflammatory damage of kidney induced by chronic intermittent hypoxia （CIH）. METHODS C57BL/6N mice were divided into normal （control） group, model （CIH） group and treatment （SDG） group. The changes of the body weight was recorded. Hematoxylin-eosin （HE） staining was used to observe the morphological alterations in the renal tissues. The levels of serum creatinine and blood urea nitrogen were measured by a biochemical analyzer. Hydroxylamine and thiobarbituric acid methods were used to detect the activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the renal tissues. The protein levels of thioredoxin-interacting protein （TXNIP） and nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） were detected by immunohistochemical staining, while those of tumor necrosis factor-α （TNF-α）, interleukin-6 （IL-6） and IL-1β were measured by ELISA. The protein levels of TXNIP, NLRP3, apoptosis-associated speck-like protein containing a CARD （ASC）, caspase-1, IL-1β and IL-18 in the renal tissues were also determined by Western blot. RESULTS No significant difference in the body weight and kidney index among the 3 groups was observed （P>0.05）. HE staining showed the swollen epithelial cells of renal tubules with vesicular degeneration, and irregular glomerular morphological change in CIH group, while SDG treatment attenuated the above changes. Compared with control group, the levels of serum creatinine, TNF-α, IL-6 and IL-1β were significantly increased in CIH group （P<0.05）. The significantly increased expression levels of NLRP3 and TXNIP in the cytoplasm of renal tubular epithelial cells in CIH group were detected by immunohistochemical staining. Compared with control group, the activity of SOD was decreased, the content of MDA was increased in CIH group, and the protein expression levels of TXNIP, NLRP3, ASC, caspase-1, IL-1β and IL-18 were up-regulated and then decreased after SDG treatment （P<0.05）. CONCLUSION SDG attenuates the renal inflammatory damage of the mice induced by CIH, and its mechanism may be associated with the inhibition of oxidative stress and activation of NLRP3 inflammasome.     

Up-regulation of HO-1 protects ADSCs under serum-free and hypoxic conditions

AIM:To investigate the protective effects of endogenous heme oxygenase 1 (HO-1) induced by cobalt protoporphyrin (Copp, a HO-1 inducer) on adipose tissue-derived stromal cells (ADSCs) under the condition of serum-free and hypoxia. METHODS:The ADSCs were isolated from SD rat and cultured. The cell apoptotic rate was detected by DAPI staining. The protein expression of HO-1, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) and cleaved caspase-1 in ADSCs was messured by Western blotting. IL-1β level in supernatant was determined by ELISA. The level of intracellular reactive oxygen species (ROS) was detected using DCFH-DA. RESULTS:The up-regulation of HO -1 was induced by CoPP in a dose dependent manner and was most significant at 20 μmol/L. The increased expression of HO-1 induced by CoPP significantly reduced the apoptotic rate of ADSCs, intracellular ROS level and IL-1β secretion, and inhibited the overexpression of NLRP3, ASC and cleaved caspase-1 under serum and oxygen deprivation. These protective effects were reversed by zinc protoporphyrin (ZnPP, an HO-1 inhibitor) given simultaneously. CONCLUSION: The up-regulation of HO -1 expression induced by CoPP plays protective effect on ADSCs under the condition of serum and oxygen deprivation via inhibiting the activation of NLRP3 inflammasome and reducing IL-1β secretion.     

苹果miR396家族鉴定及在不定根发育过程中的表达分析

分析了苹果miR396家族进化特性及其在苹果不定根发育过程中的表达模式。结果表明：苹果miR396家族有4条成熟体和7条前体序列（pre-miRNA）。Mfold预测显示Pre-miR396家族7个成员序列均可形成典型稳定的茎环二级结构,最小折叠自由能介于–62.9 kal ? mol-1（pre-miR396b）~–51.9 kal ? mol-1（pre-miR396g）之间。系统发育进化树分析显示,pre-miR396家族亲缘关系可分为3个亚组（G1、G2、G3）,每个亚组内基因数量不同,分别含有11、9、19个。靶基因预测显示,苹果miR396靶基因包括MdGRF1、MdGRF2和MdGRF5等,降解组测序进一步验证了miR396对其候选靶基因MdGRF1、MdGRF2和MdGRF5的剪切关系。苹果miR396家族成员在侧根和果实中的表达量显著高于其他组织,其候选靶基因表达量则在花芽和腋芽中显著高于其他组织;不定根发育过程中,miR396家族不同成员表达模式存在显著差异,整体上呈上调表达趋势,其候选靶基因呈下调表达趋势;外源IBA处理显著诱导miR396家族成员的表达,尤其是在不定根诱导期和根系生长期更为显著。     

LPS regulates macrophage autophagy through PI3K/Akt/mTOR pathway

AIM: To detect the activation of macrophage autophagy caused by lipopolysaccharide (LPS) and the possible related signaling pathways. METHODS: The macrophage cell line RAW264.7 cultured in vitro was divided into 5 groups according to the culture environment, including normal culture group, starvation-activated sautophagy group, LPS group, LPS+PI3K inhibitor (hVps34) group and LPS+mTOR inhibitor (rapamycin) group. Fluorescent expression vector pcDNA3.1-GFP-LC3 constructed in previous work was transfected into the macrophages.The fluorescence microscopy was used to detect the formation of autophagosome. The mRNA expression of autophagy-associated genes Atg5, Atg7, LC3-II and Bnip3 in the macrophages was detected by qRT-PCR. The protein levels of LC3-II, p-Akt and p-mTOR were determined by Western blotting, so as to evaluate the molecular pathways of autophagy in LPS-activated macrophages. RESULTS: The macrophages stably expressing GFP-LC3 were successfully established, which were used to observe the autophagy under fluorescence microscope.Compared with normal culture group, the autophagy in starvation group, LPS+hVps34 group and LPS+rapamycin group was significantly increased. The mRNA expression levels of Atg5, LC3-II and Bnip3 were significantly increased in starvation group, LPS+hVps34 group and LPS+rapamycin group, while in LPS group, those decreased slightly. The protein level of p-Akt in starvation group, LPS group and LPS+rapamycin group was significantly increased, while p-mTOR in starvation group, LPS+hVps34 group and LPS+rapamycin group significantly declined. LC3-II expression level in starvation group, LPS+hVps34 group and LPS+rapamycin group was higher than that in control group and LPS group. CONCLUSION: LPS regulates macrophage autophagy, and its possible pathway is the PI3K/Akt/mTOR pathway, but there are some other effective regulatory pathways.     

EGCG antagonizes hypoxia-induced autophagy and apoptosis in PC12 cells by mTOR regulation

AIM:Hypoxia (evoked by CoCl2)-induced apoptosis and autophagy are emerging as crucial events in the etiopathology of many neurodegenerative diseases. Epigallocatechin gallate (EGCG) is the active ingredient in tea polyphenols with abilities of anti-apoptosis and anti-autophagy, but the mechanism has not been fully elucidated. In recent years, studies have reported that the mammalian target of rapamycin (mTOR) involved in the regulation of a variety of neurological like differentiation and maturation of nerve cells, anti-oxidative stress, etc. Therefore, we investigate that whether EGCG protects PC12 from hypoxia-induced apoptosis and autophagy by enhancing mTOR expression. METHODS:The expression of mTOR and beclin-1 were detected by Western blotting. The expression of caspase-3 was measured by ELISA. The cell viability was detected by CCK-8 assay. The LC-3 expression in nucelus was observed by immunofluorescence. RESULTS:Hypoxia induced apoptosis and autophagy in PC12 cells. EGCG antagonized hypoxia-induced apoptosis and autophagy by enhancing mTOR expression. Blocking the pathway of mTOR reversed the protective effect of EGCG on PC12 cells. CONCLUSION: EGCG antagonizes hypoxia-induced autophagy and apoptosis in PC12 cells by controlling mTOR regulation.     

Corticosterone inhibits LPS-induced expression of NLRP3 in mouse macrophages and its relation with XO

AIM: To investigate the inhibitory effect of corticosterone (CORT) on lipopolysaccharide (LPS)-induced expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and its relation with xanthine oxidase (XO). METHODS: An inflammatory model of mouse macrophage RAW 264.7 was established by stimulating with LPS. Total cellular protein was extracted after the macrophages were treated with CORT at different concentrations (0~900 μg/L). The protein levels of NLRP3 and caspase-1 were determined by Western blot. According to the treatments, the macrophages were divided into control group, LPS group, LPS+CORT group and LPS+allopurinol group. Cell components were extracted at 0, 0.5, 1, 1.5 and 2 h. The protein levels of NLRP3 and XO were determined by Western blot,and the mRNA expression of NLRP3 and XO was detected by real-time PCR. RESULTS: CORT at 700 μg/L and above significantly inhibited the expression of NLRP3 and the activation of caspase-1 in the macrophages induced by LPS (P<0.05). Compared with LPS group, the expression of NLRP3 and XO in LPS+CORT group was inhibited (P<0.05), and the expression of NLRP3 in LPS+allopurinol group was also reduced (P<0.05).CONCLUSION: High concentration of CORT inhibits the expression of NLRP3 in LPS-induced mouse macrophages, which is associated with XO. The inhibitory effect of CORT may be related to the reduction of XO expression.