Effect of exogenous hydrogen sulfide on lipophagy in mouse primary hepatocytes

AIM: To evaluate the effect of exogenous hydrogen sulfide (H₂S) from GYY4137 on lipophagy in mouse primary hepatocytes. METHODS: The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion method were divided into 4 groups: the cells in control group were incubated with normal medium; the cells in model group were incubated with 1.2 mmol/L oleic acid (OA) for 48 h; the cells in H₂S group or propargylglycine (PAG) group were incubated with 1.2 mmol/L OA for 48 h followed by serum-free phenol red-free RPMI-1640 medium which contained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h. The cells were collected to conduct immunofluorescence staining of LC3 and photography under fluorescence microscope, phase-contrast microscope or transmission electron microscope. The protein expression of LC3-Ⅰ/Ⅱ in the hepatocytes was determined by Western blot. RESULTS: In contrast with the model group, the fluorescent particles of LC3, the protein expression of LC3, the number of autophagic lysosome and vacuoles in hepatocytes in H₂S group increased. CONCLUSION: In steatosis hepatocytes, exogenous H₂S promotes the lipophagy.     

Effect of GYY4137 on cytosolic lipid decomposition in mouse primary hepatocytes

AIM: To evaluate the effect of GYY4137, a novel hydrogen sulfide (H₂S) donor, on cytosolic lipid decomposition in mouse primary steatosis hepatocytes. METHODS: Oleic acid (OA) was used to induce hepatic steatosis model in vitro. The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion were divided into 4 groups:the cells in control group were incubated with normal medium for 54 h; the cells in model group were incubated with OA at 1.2 mmol/L for 48 h followed by serum-free phenol red-free RPMI-1640 for 6 h; the cells in H₂S group or DL-propar-gylglycine (PAG; an inhibitor of cystathione γ-lysase, inhibiting H₂S synthesis) group were incubated with OA at 1.2 mmol/L for 48 h followed by serum-free phenol red-free RPMI-1640 which contained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h. The glycerin release and the protein expression of hormone-sensitive lipase (HSL) in the cells were mea-sured. RESULTS: Compared with model group, the glycerin release and the protein expression of phosphorylated HSL (p-HSL) in H₂S group decreased significantly, while those increased significantly in PAG group. CONCLUSION: In steatosis hepatocytes, exogenous H₂S possibly decreases cytosolic lipid decomposition by decreasing the protein level of p-HSL.     

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.     

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.     

Role of ATP-sensitive potassium channels in inhibitory effect of hydrogen sulfide on high glucose-induced inflammation mediated by necroptosis in H9c2 cardiac cells

AIM: To investigate the role of ATP-sensitive potassium (K_ATP) channels in the inhibitory effect of hydrogen sulfide (H₂S) on high glucose(HG)-induced inflammation mediated by necroptosis in H9c2 cardiac cells.METHODS: The expression levels of receptor-interacting protein 3 (RIP3; an indicator of necroptosis) and cyclooxyge-nase-2 (COX-2) were determined by Western blot. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected by ELISA.RESULTS: After H9c2 cardiac cells were treated with 35 mmol/L glucose (HG) for 24 h, the expression of RIP3 was significantly increased. Pre-treatment of the cells with 100 μmol/L diazoxide (DZ; a K_ATP channel opener) or 400 μmol/L NaHS (a donor of H₂S) for 30 min considerably blocked the up-regulation of RIP3 induced by HG. Moreover, pre-treatment of the cells with 100 μmol/L 5-hydroxydecanoic acid (5-HD; a K_ATP channel blocker) attenuated the inhibitory effect of NaHS on HG-induced up-regulation of RIP3. On the other hand, co-treatment of the cells with 100 μmol/L necrostatin-1 (a specific inhibitor of necroptosis) or pre-treatment of the cells with 100 μmol/L DZ or 400 μmol/L NaHS attenuated HG-induced inflammatory responses, evidenced by decreases in the expression of COX-2 and secretion levels of IL-1β and TNF-α. However, pre-treatment of the cells with 100 μmol/L 5-HD significantly attenuated the above anti-inflammatory effects of NaHS.CONCLUSION: K_ATP channels play an important role in the inhibitory effect of H₂S on HG-induced inflammation mediated by necroptosis in H9c2 cardiac cells.     

Identification of ATTM as a novel H2S donor and investigation of its protective effect on HaCaT skin cells

AIM: To investigate the ability of a metal complex ammonium tetrathiomolybdate (ATTM) to release H₂S and its cytoprotective effect on an oxidative injury model. METHODS: Released H₂S was absorbed in a reaction flask from ATTM dissolved in the cell medium. Staining with dichlorodihydrofluorescein diacetate or rhodamine 123 followed by photofluorography was conducted for the observation of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ_m) levels, respectively. Cell viability and release of lactate dehydrogenase (LDH) from the cells were measured with commercial kits. RESULTS: Similar to another H₂S donor GYY4137, ATTM had an ability to release H₂S in the cell medium in a dose-dependent manner. Treatment of human skin HaCaT cells with ATTM at concentrations of 25~400 μmol/L didn't significantly alter cell viability. Exposure of the cells to ultraviolet rays or a ROS donor H₂O₂ increased the intracellular ROS levels. Treatment with 400 μmol/L H₂O₂ significantly reduced the viability of HaCaT cells (P<0.01). However, before the treatment with H₂O₂, pretreatment with ATTM at 100 and 200 μmol/L markedly prevented the H₂O₂-induced cell injury (P<0.01). In addition, the treatment with H₂O₂ triggered ΔΨ_m loss (P<0.01) and LDH release from the cells (P<0.01). Prior to suffering from H₂O₂ injury, the preconditioning with 200 μmol/L ATTM significantly improved ΔΨ_m levels (P<0.05) and attenuated LDH release from the cells (P<0.01).CONCLUSION: ATTM is capable of releasing H₂S and protecting human skin cells against oxidative injury.     

Hydrogen sulfide protects H9c2 cardiomyocytes against high glucose-induced injury by inhibiting necroptosis

AIM: To study whether hydrogen sulfide(H₂S) protects H9c2 cardiomyocytes against high glucose(HG)-induced injury by inhibiting necroptosis. METHODS: The protein levels of RIP3(an indicator of necroptosis) and cleaved caspase-3 were determined by Western blot. The cell viability was measured by CCK-8 assay. The intracellular le-vels of reactive oxygen species(ROS) were detected by 2', 7'-dichlorfluorescein diacetate staining followed by photofluorography. Mitochondrial membrane potential(MMP) was examined by rhodamine 123 staining followed by photofluorography. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography. RESULTS: After the H9c2 cells were treated with HG(35 mmol/L glucose) for 0~24 h, the protein expression of RIP3 in the H9c2 cells was significantly increased at 3 h, 6 h, 9 h, 12 h and 24 h, reaching the maximum level at 24 h. Pretreatment of the cells with 400μmol/L NaHS(a donor of H₂S) or co-treatment of the cells with necrostatin-1(Nec-1; a speci-fic inhibitor of necroptosis) considerably blocked the up-regulation of RIP3 protein induced by HG. Moreover, pretreatment with NaHS or co-treatment with Nec-1 obviously inhibited HG-induced injuries, leading to an increase in the cell viability, and decreases in the generation of ROS and MMP loss. On the other hand, pretreatment with NaHS also reduced the number of apoptotic cells and the protein level of cleaved caspase-3 in the HG-treated H9c2 cardiomyocytes. CONCLUSION: H₂S protects H9c2 cardiomyocytes against HG-induced injury by inhibiting necroptosis.     

Influence of hydrogen sulfide on ileal epithelial cell apoptosis in rats with intestinal ischemia-reperfusion injury

AIM: To investigate the influence of hydrogen sulfide (H₂S) on intestinal epithelial cell mitochondrial morphology and function and the expression of caspase-3, cleaved caspase-3, cytochrome C (Cyt C), Bcl-2 and Bax in rats with intestinal ischemia-reperfusion (I/R) injury. METHODS: Wistar rats (n=24) were randomly divided into 3 groups (8 in each group): sham group, I/R group and I/R+sodium hydrosulfide (NaHS) group. The animal model of intestinal I/R injury was established. The rats in I/R+NaHS group received NaHS (100 μmol/kg bolus +1 mg·kg^-1·h^-1 infusion) 10 min prior to the onset of reperfusion, whereas the rats in I/R group and sham group received equal volume of normal sodium. Ileum epithelial mitochondrial morphology and function were measured. Plasma H₂S was detected by sensitive sulfide electrode. The expression of Bcl-2 and Bax mRNA was studied by RT-PCR. The protein levels of caspase-3, cleaved caspase-3, cytochrome C (Cyt C), Bcl-2 and Bax were tested by Western blot.RESULTS: The area, volume density, maximum diameter, minimum diameter and equivalent diameter of mitochondria, and the expression of cleaved caspase-3, Cyt C and Bax in I/R group were significantly higher than those in I/R+NaHS and sham groups (P<0.01). The mitochondrial count, circumference, specific surface area, area density and population density, plasma H₂S, respiratory control rate (RCR), the ratio of P/O, R₃ , R₄, and the expression of Bcl-2 in I/R group were sharply lower than those in I/R+NaHS and sham groups (P<0.01). H₂S was negatively correlated with caspase-3, cleaved caspase-3, Cyt C and Bax (P<0.01), and was positively correlated with Bcl-2 (P<0.01). CONCLUSION: H₂S has a protective effect on mitochondrial morphology and function in rats with intestinal I/R injury by down-regulating cleaved caspase-3, Cyt C and Bax and up-regulating Bcl-2.     

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.     

Role of ATP-sensitive potassium channels in inhibitory effect of hydrogen sulfide on high glucose-induced injury in H9c2 cardiac cells

AIM: To investigate the roles of ATP-sensitive potassium (K_ATP) channels in high glucose-induced cardiac injury and the inhibitory effect of hydrogen sulfide (H₂S) on the cardiomyocyte injury. METHODS: The expression level of K_ATP channel protein was tested by Western blot. The cell viability was measured by CCK-8 assay. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining. Mitochondrial membrane potential (MMP) was examined by JC-1 staining. RESULTS: After the H9c2 cells were treated with 35 mmol/L glucose (high glucose, HG) for 1~24 h, the protein level of K_ATP channel was significantly reduced at 6 h, 9 h, 12 h and 24 h, reaching the minimum level at 12 h and 24 h. Pretreatment of the cells with 400 μmol/L NaHS (a donor of H₂S) prior to exposure to HG for 12 h considerably blocked the down-regulation of K_ATP channels induced by HG. Pretreatment of the cells with 100 μmol/L mitochondrial K_ATP channel opener diazoxide, 50 μmol/L non-selective K_ATP channel opener pinacidil or NaHS obviously inhibited HG-induced injuries, leading to an increase in the cell viability, and decreases in the number of apoptotic cells and the MMP loss. Pretreatment with 100 μmol/L mitochondrial K_ATP channel antagonist 5-hydroxydecanoic acid or 1 mmol/L non-selective K_ATP channel antagonist glibenclamide attenuated the above cardioprotective effects of NaHS. CONCLUSION: K_ATP channels mediate the inhibitory effect of H₂S on HG-induced cardiac injury.     

Protective effect of ecdysterone on H9c2 cells against oxidative stress

AIM: To investigate the effect of ecdysterone (EDS) on H9c2 cardiomyocytes after oxidative stress. METHODS: H9c2 cells were cultured in vitro and divided into control group, high dose (2 μmol/L) of EDS group, middle dose (1.5 μmol/L) of EDS group, low dose (1 μmol/L) of EDS group, and H₂O₂ group. H9c2 cardiomyocytes in H₂O₂ group and high, middle and low doses of EDS groups were exposed to H₂O₂ for 6 h to establish the model of oxidative stress. The viability of the H9c2 cells was detected by CCK-8 assay. The apoptosis of H9c2 cells was analyzed by flow cytometry. The levels of lactate dehydogenase (LDH) and creatine kinase-MB (CK-MB) in the culture medium, and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in the H9c2 cells were measured by colorimetry. The generation of reactive oxygen species (ROS) and the mitochondrial membrane potential were evaluated by flow cytometry and confocal laser scanning microscopy. The protein levels of Bax, Bcl-2 and cleaved caspase-3 in the H9c2 cells were determined by Western blot. RESULTS: Ecdysterone at the selected concentrations had no effect on the viability of H9c2 cells. Compared with control group, the levels of LDH, CK-MB, ROS and MDA, and the apoptotic rates of the H9c2 cells were significantly increased after treated with H₂O₂, but were decreased by EDS treatment in a dose-dependent manner. The levels of SOD and mitochondrial membrane potential of the H9c2 cells in H₂O₂ group were reduced significantly compared with control group, but high, middle and low doses of EDS treatments up-regulated the levels of SOD and mitochondrial membrane potential in H₂O₂-treated H9c2 cells. The protein levels of Bax and cleaved caspase-3 in the H9c2 cells in H₂O₂ group showed significant elevation in comparison with control group, and the protein expression of Bcl-2 declined in H₂O₂ group compared with control group, but high, middle and low doses of ecdysterone treatments down-regulated the protein levels of Bax, cleaved caspase-3 and up-regulated the expression of Bcl-2 in H₂O₂-treated H9c2 cells. CONCLUSION: Ecdysterone attenuates the effect of H₂O₂-induced oxidative stress on H9c2 cardiomyocytes. The mechanism may be involved in scavenging oxidative stress products, increasing antioxidant enzyme activity and improving mitochondrial function.     

Protective effect of senegenin on retinal ganglion cells in oxidative stress induced injury

AIM: To evaluate the effect of senegenin (Sen) on H₂O₂-treated retinal ganglion cells (RGCs) and to explore its underlying mechanisms. METHODS: RGCs were retrograde labeled by injection of fluorogold into the superior colliculi of SD rats on the postnatal day 3. On the postnatal days 6 to 8, the retinas were dissociated with papain and cultured. Primary RGCs cultured in vitro were treated with H₂O₂ and/or various doses of Sen. The viability of RGCs was evaluated by counting the fluorescence-labeled neurons under microscope. The morphological changes of the nuclei in the retinal neurons were observed by Hoechst 33258 staining. Western blotting was applied to determine the expression of cleaved caspase-3, cytochrome C and Bcl-2 in cultured retinal neurons. RESULTS: Compared with the control cells, Sen at doses of 10, 20 or 40 μmol/L had no toxicity to RGCs (P>0.05). However, Sen at doses of 80 and 160 μmol/L had significant toxicity to RGCs (P<0.01). Compared with H₂O₂-injured group, Sen at doses of 10, 20 and 40 μmol/L effectively protected against H₂O₂-induced injury in RGCs (P<0.05) with the best efficiency at 40 μmol/L. Hoechst 33258 staining showed that the neuronal apoptosis caused by H₂O₂ was reduced by Sen. The results of Western blotting showed an up-regulation of Bcl-2, and decreased cytochrome C and cleaved caspase-3 levels by Sen in H₂O₂-treated retinal neurons. CONCLUSION: Sen is able to protect RGCs from H₂O₂-induced injury by enhancing Bcl-2 expression and inhibiting cell apoptosis.     

Involvement of 26S proteasome LMP2 subunit in development of tolerance against oxidative stress in vascular endothelial cells

AIM: To observe the expression of 26S proteasome LMP2 subunit in vascular endothelial cells (VECs) under oxidative stress, and to evaluate its role in the development of tolerance against oxidative stress in VECs. METHODS: The cell model of H₂O₂ preconditioning-induced oxidative tolerance was established in VECs. The expression of LMP2 was detected by cellular immunofluorescent labeling and Western blotting. The LMP2 anti-sense and sense oligonucleotides were transfected into VECs by Lipofectamine^TM 2000. The damages of VECs were evaluated by detecting the activity of lactate dehydrogenase (LDH) and the concentration of malondialdehyde (MDA) in the culture medium. RESULTS: H₂O₂ (500 μmol/L for 3 h) induced oxidative stress in VECss in a dose- and the activity of time-dependent manner, characterized by the increase in the concentration of MDA and LDH in the culture medium. Pretreatment with H₂O₂ (10 μmol/L for 24 h) up-regulated the expression of LMP2. Meanwhile, the capacity of cellular tolerance against oxidative stress induced by H₂O₂ was increased as the concentration of MDA and the activity of LDH in the culture medium significantly decreased. Compared with H₂O₂ group, up-regulation of LMP2 by IFN-γ pretreatment (20 μg/L for 48 h) increased the tolerance of VECs against H₂O₂ injury, and the MDA conentration and the activity of LDH in the culture medium also significantly decreased. Transfection with LMP2 antisense oligonucleotide partly inhibited the increased expression of LMP2 induced by IFN-γ in VECs and abolished the tolerance against H₂O₂. CONCLUSION: The 26S proteasome LMP2 subunit is associated with the development of the tolerance against H₂O₂-induced oxidative stress in VECs.     

Acetyl-L-carnitine attenuates H2O2-induced oxidative damage of PC12 cells

AIM: To investigate the effect of acetyl-L-carnitine (ALC) on H₂O₂-induced oxidative damage in PC12 cells and its possible mechanism. METHODS: A moderate oxidative damage PC12 cell model was induced by exposure of the PC12 cells to H₂O₂. ALC at different concentrations (100, 200 and 400 μmol/L) was applied to the PC12 cells cultured in vitro, and CCK8 assay was used to detect the cell viability. The cells were divided into control group, H₂O₂ group, and low-ALC, medium-ALC and high-ALC groups. The apoptosis of the cells was analyzed by flow cytometry. The protein levels of Nrf2 and cleaved caspase-3 were determined by Western blot. The nuclear translocation of Nrf2 was observed by immunofluorescence staining. RESULTS: ALC at different concentrations (100, 200 and 400 μmol/L) significantly inhibited H₂O₂-induced PC12 cell apoptosis, and the medium concentration group had the best effect. Compared with H₂O₂ group, low, medium and high concentrations of ALC significantly increased the viability of the PC12 cells induced by H₂O₂, inhibit cell apoptosis (P<0.05), significantly down-regulated the protein level of cleaved caspase-3 (P<0.05), up-regulated the protein level of Nrf2 (P<0.05), and promoted the translocation of Nrf2 from the cytoplasm to the nucleus. CONCLUSION: Acetyl-L-carnitine attenuates H₂O₂-induced oxidative damage of PC12 cells, inhibits the apoptosis and increases the viability, which is related to the activation of Nrf2 signaling pathway.     

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.     

Endogenous hydrogen sulfide regulates oxidant/antioxidant balance in rat with chronic obstructive pulmonary disease

AIM: To investigate the role of hydrogen sulfide (H₂S) in the protection against oxidative stress in rats with chronic obstructive pulmonary disease (COPD).METHODS: The rat model of COPD was established by cigarette smoking (CS) combined with lipopolysaccharide (LPS) instillation. Thirty-two healthy male Sprague-Dawley rats were randomly divided into 4 groups: control group, CS+LPS group, CS+LPS+NaHS (H₂S donor) group and CS+LPS+PPG (DL-propargylglycine, an inhibitor of cystathionine-γ-lyase) group. After 30 days, the lung functions of the rats were measured, the histological changes of lungs were observed under light microscope and the pathological scores were calculated. The H₂S level in plasma and the protein expression of cystathionine-γ-lyase (CSE) in the lung tissues were measured. The content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and catalase (CAT) were detected to reflect oxidative stress.RESULTS: Compared with control group, the peak expiratory flow (PEF) decreased by 24% and intra-pressure (IP) increased by 66% in CS+LPS group. The pathological scores of the lung tissues also increased. Compared with CS+LPS group, no change in the lung function was observed after given NaHS or PPG, but the pathological scores decreased in CS+ LPS+ NaHS group. Compared with control group, the content of H₂S in plasma was increased by 26% on day 16. Compared with CS+LPS group, the content of H₂S in plasma of CS+LPS+PPG group was decreased by 22% after 30 days. Compared with control group, the protein expression of CSE increased, and no statistical difference among CS+LPS group, CS+ LPS+ NaHS group and CS+LPS+PPG group was observed. Compared with control group, MDA content in the lung tissues was increased by 24% in CS+LPS group, the activity of SOD was increased by 47% and the activity of CAT was increased by 52%. Compared with CS+LPS group, the MDA content in CS+LPS+NaHS group was decreased by 21%, and no statistical difference in the activity of SOD and CAT was observed. The activity of SOD decreased by 33% after given PPG.CONCLUSION: H₂S plays a role as anti-oxidant in the rats with COPD. The CSE/ H₂S pathway may be involved in the development of COPD.     

Quercitrin promotes apoptosis of gastric cancer cell line SGC7901 by inhibiting PI3K/AKT signaling pathway

AIM: To investigate whether quercitrin induces apoptosis of gastric cancer cell line SGC7901 by inhibition of PI3K/AKT signaling pathway. METHODS: The human gastric cancer SGC7901 cells were selected as the research object. The cytotoxicity of quercitrin was detected by MTT assay, and IC₅₀ value of quercitrin was calculated. The SGC7901 cells were divided into control group, quercitrin group (incubated with 200 μmol/L quercitrin), insulin-like growth factor-1 (IGF-1) group (incubated with 100 μg/L IGF-1) and quercitrin+IGF-1 group (incubated with 200 μmol/L quercitrin and 100 μg/L IGF-1). After 48 h, the apoptosis of SGC7901 cells was analyzed by flow cytometry, and the protein levels of cleaved caspase-3, p-AKT (Ser473), AKT, p-PI3K (Tyr508) and PI3K were determined by Western blot. RESULTS: The viability of SGC7901 cells was significantly decreased as the concentration of quercitrin increased, starting at 100 μmol/L (P<0.05). The IC₅₀ value of quercitrin for 48 h was 275.40 μmol/L. After treatment with 200 μmol/L quercitrin for 48 h, the apoptosis rate and the protein level of cleaved caspase-3 in quercitrin group were significantly increased (P<0.05), and the phosphorylated levels of AKT and PI3K were significantly decreased compared with control group (P<0.05). Treatment with quercitrin and IGF-1 inhibited the effect of quercitrin on SGC7901 cells compared with quercitrin group. CONCLUSION: Quercitrin may induce apoptosis of gastric cancer cell line SGC7901 by inhibiting the activation of PI3K/AKT signaling pathway.     

Effects of 6-gingerol on apoptosis of rat nucleus pulposus cells

AIM: To study the effect of 6-gingerol on the apoptosis of rat nucleus pulposus cells and its possible mechanism. METHODS: Rat nucleus pulposus cells were isolated and cultured. The effects of 6-gingerol and hydrogen peroxide (H₂O₂) at different concentrations on the viability of nucleus pulposus cells were measured by CCK-8 assay. After 6-gingerol treatment, the protein level of p-Akt was determined by Western blot. The cells were divided into 4 groups:control group, H₂O₂ group, 6-gingerol group (6-gingerol + H₂O₂) and LY294002 group (6-gingerol + H₂O₂ + LY294002). The apoptotic rate and the levels of reactive oxygen species (ROS) were analyzed by flow cytometry. TUNEL fluorescence staining was used to observe the number of apoptotic cells. The morphological changes of mitochondria were observed under transmission electron microscope, and Western blot was used to determine the protein levels of caspase-3, Bcl-2, Bax, p-Akt, Akt and p53. The mRNA expression of aggrecan and type II collagen was measured by RT-qPCR. RESULTS: The results of CCK-8 assay showed that the optimal concentration of 6-gingerol for promoting the viability of rat nucleus pulposus cells was 24 mg/L, and the exposure condition of H₂O₂ at 80 μmol/L for 6 h was appropriate for establi-shing the cell damage model. 6-Gingerol increased the protein level of p-Akt in a time-dependent manner. The apoptotic rate, ROS level and TUNEL positive cells in H₂O₂ group were significantly increased compared with control group. The mitochondrial edema was obvious in H₂O₂ group compared with control group. The protein levels of pro-apoptotic molecules caspase-3, Bax and p53 were significantly increased, while anti-apoptotic protein Bcl-2, and mRNA expression of aggrecan and type II collagen were significantly decreased compared with control group (P<0.05). 6-Gingerol exerted a protective effect against H₂O₂-induced apoptosis and promoted the expression of anti-apoptotic proteins. However, this effect was weakened after treatment with PI3K/Akt signaling pathway inhibitor LY294002. CONCLUSION: H₂O₂ induces damage and dysfunction of rat nucleus pulposus cells, and 6-gingerol may inhibit H₂O₂-induced apoptosis of nucleus pulposus cells by activation of PI3K/Akt signaling pathway.