Role of NOX1 in TNF-α-induced oxidative damage and inflammation in alveolar epithelial cells

AIM: To explore the role of NADPH oxidase 1 (NOX1) in tumor necrosis factor-α (TNF-α)-induced oxidative damage and inflammation in alveolar epithelial cells.METHODS: The mRNA and protein expression levels of NOX1 in alveolar epithelial cells after TNF-α treatment were determined by real-time PCR and Western blot. NOX1 siRNA and its negative control were transfected into the alveolar epithelial cells. After the induction of TNF-α, NOX1 levels in the cells were measured by real-time PCR and Western blot, and the content of malondialdehyde (MDA) in the cells was detected by thiobarbituric acid method. Xanthine oxidation assay was used to detect the activity of superoxide dismutase (SOD) in the cells. The contents of interleukin-4 (IL-4), IL-6 and IL-1β in cell culture medium were examined by ELISA. The rate of apoptosis was analyzed by flow cytometry. Western blot was used to detect the level of apoptotic protein cleaved caspase-3.RESULTS: The expression of NOX1 at mRNA and protein levels in TNF-α-induced cells was increased after induction (P<0.05). After transfection of NOX1 siRNA, the expression of NOX1 at mRNA and protein levels in the cell was downregulated (P<0.05). Transfection of siRNA negative control had no effect on the expression level of NOX1 in the cells. The content of MDA in the cells after TNF-α treatment was increased, the activity of SOD was reduced, the releases of IL-4, IL-6 and IL-1β by the cells were increased, and the apoptotic rate and the level of apoptotic protein cleaved caspase-3 were increased as compared with the cells that were not treated with TNF-α (P<0.05). The content of MDA in the cells with NOX1 knockdown induced by TNF-α was reduced, the activity of SOD elevated, and the releases IL-4, IL-6 and IL-1β, the apoptotic rate and the level of apoptotic protein cleaved caspase-3 decreased, as compared with the cells only treated with TNF-α induction (P<0.05).CONCLUSION: TNF-α induces the expression of NOX1 in the alveolar epithelial cells. Knockdown of NOX1 expression reduces cellular oxidative damage, releases of inflammatory factors, and cell apoptosis.     

Effect of fucoidan on intestinal ischemia-reperfusion injury in rats

AIM:To investigate the effect and potential mechanism of fucoidan on intestinal ischemia-reperfusion (I/R) injury in rats. METHODS:Adult male Wistar rats were randomly divided into 3 groups:sham group, I/R group and Fucoidan+I/R group. Fucoidan at 160 mg/kg was intraperitoneally injected in rats of Fucoidan+I/R group 7 d prior to operation, and the equal volume of saline was intraperitoneally injected in rats of sham group and I/R group. The rats in I/R group and Fucoidan+I/R group underwent superior mesenteric artery occlusion for 1 h and then reperfusion for 2 h. Following reperfusion, the histomorphological changes of the ileum were examined by HE staining. The levels of diamine oxidase (DAO), D-lactic acid (D-LA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β were detected in the blood samples, the levels of malondialdehyde (MDA) and glutathione (GSH), the activity of superoxide dismutase (SOD) and myeloperoxidase (MPO), and the protein levels of Bax, cleaved caspase-3 and Bcl-2 were analyzed in intestinal tissue samples. RESULTS:Compared with sham group and Fucoidan+I/R group, the serum levels of DAO, D-LA, TNF-α, IL-6 and IL-1β were significantly increased in I/R group (P<0.05), Chiu's score of intestinal tissue, MDA content, MPO activity, the levels of Bax and cleaved caspase-3 protein in the intestinal tissues were also significantly increased (P<0.05), while the tissue GSH content, SOD activity, and Bcl-2 protein levels were significantly decreased (P<0.05). CONCLUSION:Fucoidan attenuates intestinal tissue damage caused by I/R, which may be related to anti-oxidation, anti-inflammatory and anti-apoptotic effects.     

Effects of PTEN on apoptosis,oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation

AIM: To investigate the effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) on the apoptosis, oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation (A/R). METHODS: The H9c2 cells were used to establish a model of A/R. The H9c2 cells were transfected with PTEN small interfering RNA (siRNA) and negative control. After A/R, the expression of PTEN at mRNA and protein levels was determined by RT-PCR and Western blot, respectively. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Xanthine oxidase method was used to determine superoxide dismutase (SOD) activity. The content of malondialdehyde (MDA) was detected by thiobarbituric acid method. The lactate dehydrogenase (LDH) activity in the supernatant was evaluated by 4-dinitrophenylhydrazine method. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 in culture supernatant were examined by ELISA. The protein levels of cleaved caspase-3, Bax and FasL in the cells were determined by Western blot. RESULTS: After A/R, the expression of PTEN at mRNA and protein levels was significantly increased in the H9c2 cells (P<0.05). The mRNA and protein levels of PTEN were decreased significantly after transfection with PTEN siRNA (P<0.05). The viability of H9c2 cells was decreased after A/R, while the apoptotic rate was increased. The protein levels of cleaved caspase-3, Bax and FasL were increased in the cells. The MDA level was elevated, the activity of SOD was decreased, and the levels of LDH, TNF-α, IL-1β and IL-6 in the culture supernatant were increased (P<0.05). Down-regulation of PTEN partly antagonized the effects of A/R on the viability, apoptotic rate, MDA content, SOD activity, and the levels of LDH, TNF-α, IL-1β and IL-6 in culture supernatant. CONCLUSION: Down-regulation of PTEN attenuates oxidative damage induced by A/R, reduces apoptosis and secretion levels of TNF-α, IL-1β and IL-6 in the H9c2 cells.     

Effect of netrin-1 on high glucose induced injury of renal tubular epithe-lial cells

AIM:To study the effect of netrin-1 on the damage of renal tubular epithelial cells induced by high glucose. METHODS:Human renal tubular epithelial HK-2 cells were treated with high glucose. Real-time PCR and Western blot were used to detect the expression level of netrin-1 in the cells. HK-2 cells were infected with netrin-1-over-expressing lentivirus, and the effect of netrin-1 over-expression on the HK-2 cells treated with high glucose was observed. The apoptosis rate was analyzed by flow cytometry. The protein level of cleaved caspase-3 was determined by Western blot. lactate dehydrogenase (LDH) activity in the culture medium was measured by 2,4-binitrobenzene hydrazine method. The content of malondialdehyde (MDA) in the culture medium was detected by thiobarbituric acid method. The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the culture medium were measured by ELISA. RESULTS:The expression of netrin-1 at mRNA and protein levels in the HK-2 cells after high glucose treatment was significantly lower than that in the control cells (P<0.05). Infection with netrin-1-over-expressing lentivirus up-regulated the expression of netrin-1 in the HK-2 cells treated with high glucose. High glucose promoted the secretion of IL-1β and TNF-α, decreased the levels of LDH and MDA in the cell culture supernatant, and induced apoptosis and activation of caspase-3 in renal tubular epithelial cells (P<0.05). After the HK-2 cells with up-regulation of netrin-1 were induced by high glucose, the IL-1β and TNF-α secretion, the levels of LDH and MDA in the culture medium, the apoptosis, and the level of activated caspase-3 protein in the cells were all decreased, as compared with the control cells (P<0.05). CONCLUSION:Up-regulation of netrin-1 expression attenuates oxidative damage and inflammatory injury, and reduces apoptosis induced by high glucose in renal tubular epithelial cells.     

Effects of procyanidins on oxidative damage of osteocytes caused by TCP wear particles

AIM: To investigate the effects of procyanidins (PC) on oxidative damage of osteocytes caused by tricalcium phosphate (TCP) wear particles, and to explore the underling mechanism. METHODS: Mouse long bone osteocyte MLO-Y4 cells were treated with TCP wear particles (0.1 g/L) for 48 h to establish the model of osteocyte injuries. The MLO-Y4 cells were divided into 4 groups:control group, TCP group, PC (10 μmol/L) group and PC (50 μmol/L) group. Calcein-AM staining and MTT assay were used to observe the viability of MLO-Y4 cells. The levels of dentin matrix protein 1 (DMP-1), sclerostin (SOST) and interleukin-1β (IL-1β) in the culture media were examined by ELISA. The apoptosis of MLO-Y4 cells was analyzed by flow cytometry with Annexin V/PI double staining. The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity of MLO-Y4 cells, and lactate dehydrogenase (LDH) release in the culture media were measured by chemical colorimetry. The protein levels of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), cleaved caspase-1 and IL-1β in the MLO-Y4 cells were determined by Western blot. RESULTS: Compared with control group, MLO-Y4 cell injuries, apoptosis rate and MDA level were obviously increased in TCP group, while SOD activity was significantly decreased (P<0.05) The protein levels of NLRP3, ASC, cleaved caspase-1 and IL-1β were remarkably up-regulated (P<0.05) in the MLO-Y4 cells, and the level of IL-1β and LDH release were increased in the culture media (P<0.05). Compared with TCP group, the injuries of MLO-Y4 cells, apoptosis rate and MDA level were decreased obviously (P<0.05) in PC groups, whereas SOD activity was increased (P<0.05). The protein levels of NLRP3, ASC, cleaved caspase-1 and IL-1β were down-regulated remarkably in the MLO-Y4 cells (P<0.05), and the level of IL-1β and LDH release were significantly decreased in the culture media (P<0.05). CONCLUSION: PC obviously inhibit oxidative damage of osteocytes caused by TCP wear particles, which may be related to alleviating NLRP3 inflammasome activation and pyroptosis.     

Poly(adenosine diphosphate ribose) glycohydrolase regulates apoptosis-inducing factor and inflammatory factors in rat hippocampus after seizures

AIM: To investigate the effects and molecular mechanisms of poly(adenosine diphosphate ribose) glycohydrolase (PARG) on rat hippocampus neurons after seizures and to study the effects of gallotannin on the expression of apoptosis-inducing factor (AIF), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in rat hippocampus after seizures. METHODS: Seizures were induced by kainic acid (KA). The damage of hippocampus neurons was evaluated by Nissl staining. The protein expression levels of poly(adenosine diphosphate ribose) (PAR), AIF, IL-1β and TNF-α were detected by Western blotting analysis. RESULTS: The number of damaged hippocampal pyramidal neurons in gallotannin-treated group was significantly lower than that in KA-treated group (P<0.05). The expression of PAR was increased in gallotannin-treated group compared with KA-treated group(P<0.05).AIF in mitochondrial fraction increased and its accumulation in nucleus fraction decreased in gallotannin-treated group compared with KA-treated group (P<0.05). In addition, gallotannin significantly decreased the protein expression of IL-1β and TNF-α, which were obviously increased in hippocampus after seizures (P<0.05). CONCLUSION: PARG inhibition by gallotannin has the neuroprotective effect on the damage of hippocampal neurons induced by seizures. In addition, gallotannin suppresses the translocation of AIF from mitochondria to nucleus and the expression of IL-1β and TNF-α in rat hippocampus after seizures.     

Pretreatment with external trigeminal nerve electrostimulation inhibits seizures and decreases expression of IL-1β and TNF-α in hippocampus with status epilepticus induced by pentylenetetrazol in rats

AIM:To observe the effect of pretreatment with external trigeminal nerve electrostimulation (eTNS) on behavioral changes and the expression of interleukin-1β (IL-1β) and 　tumor necrosis factor α (TNF-α) in hippocampus of pentylenetetrazol (PTZ)-treated rats. METHODS:The rats were randomly divided into control group, PTZ group and eTNS group, and kindled by PTZ after administered 7 d, 14 d and 28 d of consecutive fake electrostimulation or eTNS. Subsequently, the severity and duration of seizure were quantitatively evaluated. The concentrations of IL-1β and TNF-α in hippocampus were detected by the methods of ELISA and immunohistochemisty. RESULTS:Compared with PTZ group, treatment with eTNS significantly inhibited the severity and duration of seizure (P<0.05), and significantly reduced the content of IL-1β and TNF-α in the hippocampus after status epilepticus (P<0.05 or P<0.01). CONCLUSION:Pretreatment with eTNS may provide a new approach for prevention and treatment of epileptogenesis.     

Preliminary study on pyroptosis involved in cerebral ischemia-reperfusion injury

AIM:To investigate the changes of pyroptosis in hippocampus and cortex at different time points after cerebral ischemia-reperfusion, and to explore its mechanism from NLRP3-mediated classical pyroptosis pathway, and to analyze the role of pyroptosis in different parts of cerebral injury. METHODS:SD rats were randomly divided into sham operation group (sham group) and model group (MCAO/R group). The rats in model group was further divided into cerebral ischemia-reperfusion 6 h group (MCAO/R 6 h group), 12 h group (MCAO/R 12h group)and 24 h group (MCAO/R 24 h group). The rat model was established on rats by middle cerebral artery occlusion and reperfusion (MCAO/R) induced by modified right-side thread method. Neurologic function score, 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and morphological observation were used to evaluate the degree of nervous cell injury. TUNEL and caspase-1 immunofluorescence double staining were used to detect pyroptosis. The protein expression of NLRP3, cleaved caspase-1, pro-caspase-1 and interleukin-1β (IL-1β) was determined by Western blot. RESULTS:Neurological damage occurred at different times after cerebral ischemia-reperfusion. TTC staining showed that the volume of cerebral infarction gradually increased with the prolongation of reperfusion time (P<0.05). The hippocampal CA1 area and cortical area showed typical morphological features such as loose tissue structure, interstitial edema, disordered arrangement of nerve cells, deepening of nucleus staining, nuclear fragmentation and decreased cell number. Immunofluorescence double staining showed that there was a phenomenon of pyroptosis at different time after cerebral ischemia-reperfusion. The pyroptosis of hippocampal CA1 and cortical area was most obvious at 12 h and 24 h after reperfusion (P<0.05). Western blot analysis showed that the expression of NLRP3, cleaved caspase-1, pro-caspase-1 and IL-1β in NLRP3-mediated classic pyroptosis pathway was regulated in different degrees after cerebral ischemia-reperfusion. The protein expression of NLRP3 in hippocampus was significantly increased at 12 h and 24 h after reperfusion (P<0.05), and the protein expression of NLRP3 in cortex was significantly increased at 6 h after reperfusion (P<0.05). The protein expression of pro-caspase-1 in hippocampus was significantly increased at each time points of reperfusion (P<0.05), and the protein expression of pro-caspase-1 in the cortex was significantly increased at 24 h after reperfusion (P<0.05). The protein expression of cleaved caspase-1 in the hippocampus was significantly increased at 12 h after reperfusion (P<0.05), and increased in the cortex at 24 h after reperfusion (P<0.05). The protein expression of IL-1β in the hippocampus was significantly increased at 24 h after reperfusion (P<0.05), and increased in the cortex at 6 h after reperfusion (P<0.05). CONCLUSION:Pyroptosis is involved in neuronal injury after cerebral ischemia-reperfusion. The classic pyroptosis pathway plays an important regulatory role in hippocampus and cortex, especially in hippocampus, suggesting that hippocampus is the main part of secondary nerve impairment induced by pyroptosis and inflammation after cerebral ischemia-reperfusion.     

Heme oxygenase-1 protects renal function in septic rats via influencing expression of thrombomodulin

AIM: To investigate the protective effect of heme oxygenase-1 (HO-1) on the kidney of septic rats and the influence of HO-1 on the expression of thrombomodulin (TM) in the kidney. METHODS: Sepsis in rats was developed with cecal ligation and puncture (CLP). The septic rats were randomly divided into sham group, CLP group, CLP+HO-1 inducer group and CLP+HO-1 inhibitor group (n=18). The plasma levels of creatinine (Cr), cystatin-C (Cys-C), carboxyhemoglobin (COHb), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and TM, and the changes of prothrombin time (PT) and activated partial thromboplastin time (APTT) in each group were measured. Histopathological examination was performed in the kidney. The expression of TM in the kidney tissue was detected by Western blot. RESULTS: Compared with sham group, significantly elevated plasma levels of Cr, Cys-C, TNF-α, IL-1β and TM (P<0.05), shortened PT and APTT (P<0.05), significantly increased microthrombus formation, and lowered TM expression in the kidney (P<0.05) of CLP group were observed. The administration of hemin lowered the plasma levels of Cr, Cys-C, TNF-α, IL-1β and TM (P<0.05), prolonged PT and APTT (P<0.05), attenuated microthrombus formation, and up-regulated the expression of TM in the kidney (P<0.05). In contrast, ZnPP had the opposite effects. CONCLUSION: HO-1 increases the expression of TM in the kidney and exerts anticoagulatory and antiinflammatory effects, thereby improving renal function in the septic rats.     

Effect of NOD8 on production of nitric oxide,IL-1β and TNF-α in LPS-treated macrophages

AIM: To investigate the effect of NOD8 on lipopolysaccharide (LPS)-induced releases of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 cells. METHODS: The plasmids of pEGFP-C2 and pEGFP-NOD8 were transfected into RAW264.7 cells respectively. The transfected and non-transfected cells were stimulated by LPS for 0, 6, 12 and 24 h. NO production was evaluated by Griess reagent assay, and the levels of IL-1β and TNF-α were measured by ELISA. The protein expression of NOD8 and the nuclear translocation of nuclear factor κB (NF-κB) p65 subunit were detected by Western blotting. The level of activated caspase-1 was determined by fluorimetric method. RESULTS: Compared with pEGFP-C2 group, the protein expression of NOD8 was significantly elevated in pEGFP-NOD8+LPS group. The releases of NO, IL-1β and TNF-α were obviously increased after RAW264.7 cells were treated with LPS for 6 h, 12 h and 24 h, and while the secretion of NO was significantly reduced in the cells transfected with pEGFP-NOD8 and induced by LPS for 12 h and 24 h, and the release of IL-1β was also significantly reduced at 6 h, 12 h and 24 h. However, no significant difference of TNF-α release was observed between pEGFP-C2+LPS group and pEGFP-NOD8+LPS group. The activation of caspase-1 in RAW264.7 cells stimulated with LPS for 6 h, 12 h and 24 h was markedly increased, and the expression of NF-κB p65 subunit in the cytoplasm was significantly decreased, indicating that p65 nuclear translocation was increased. In addition, the activation of caspase-1 and the nuclear translocation of p65 were significantly inhibited in pEGFP-NOD8+LPS group. CONCLUSION: NOD8 suppresses the releases of LPS-induced NO and IL-1β in RAW264.7 cells by inhibiting the activation of caspase-1 and NF-κB.     

Therapeutic effect of Jiawei Danzhi Xiaoyao powder combined with intravenous human umbilical cord mesenchymal stem cells on post-stroke depression

AIM: To observe the clinical therapeutic effect of Jiawei Danzhi Xiaoyao powder combined with transplantation of human umbilical cord mesenchymal stem cells (HUC-MSCs) in the treatment of post-stroke depression (PSD) patients and to detect the changes of the serum cytokine levels of the tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, norepinephrine (NE), 5-hydroxytry-ptamine (5-HT) and brain derived neurotrophic factor (BDNF). METHODS: The patients with PSD (n=60) were randomly divided into observation group and control group. The patients in observation group was given Jiawei Danzhi Xiaoyao powder combined with HUC-MSCs, and the patients in control group was given Jiawei Danzhi Xiaoyao powder combined with fluoxetine. The course of treatment was 8 weeks. The effects of the treatments on the patients were evaluated with Hamilton Depression Scale (HAMD). The serum levels of IL-1β, IL-6, TNF-α, 5-HT, NE and BDNF were measured by ELISA. RESULTS: After 8 weeks of treatment, the total effective rate in observation group was significantly higher than that in control group (P<0.05). Compared with the same group before treatment, the HAMD scores and the serum levels of TNF-α, IL-1β, IL-6 of the 2 groups were significantly decreased after treatment (P<0.05), while 5-HT, NE and BDNF were significantly increased (P<0.05). After 8 weeks of treatment, the HAMD scores in observation group was significantly lower than that in control group (P<0.05), and the serum levels of TNF-α, IL-1β, IL-6 were significantly decreased in observation group (P<0.05), while 5-HT, NE and BDNF were significantly increased (P<0.05). CONCLUSION: Jiawei Danzhi Xiaoyao powder combined with human umbilical cord mesenchymal stem cells is effective in the treatment of post-stroke depression. The mechanism may be related to the effects of HUCMSCs such as anti-inflammatory effect, increased release of monoamine neurotransmitters, and stimulation of secretion of neurotrophic factors in the brain.     

Effect of GRK5 on activation of rat astrocytes

AIM: To study the effect of G-protein-coupled receptor kinase 5 (GRK5) on the activation of astrocytes in the brain cortex of newborn Wistar rats. METHODS: GRK5 gene was silenced in the model of rat brain cortex astrocytes in vitro for 24 h. N-acetylcysteine (NAC), which is a known inhibitor of NF-κB, was added into the culture medium according to gene silencing for 24 h. The expression levels of GFAP and caspase-3 were detected by the method of immunofluorescence, and the mRNA levels of NF-κB, TNF-α, IL-1β and iNOS were determined by real-time PCR. Moreover, the activity of SOD and concentrations of TNF-α and NO were measured. RESULTS: GRK5 gene silencing increased the expression of NF-κB at mRNA and protein levels obviously (P<0.01), and the mRNA levels of IL-1β and iNOS increased synchronously (P<0.01). Furthermore, caspase-3-positive cells in GRK5 siRNA group were increased compared with control siRNA group (P<0.01). Treatment with NAC obviously reduced the activity of NF-κB and weakened the effects induced by GRK5 siRNA (P<0.05). CONCLUSION: GRK5 siRNA increases NF-κB activity and induces the activation of astrocytes.     

Effects of amifostine on formation of abdominal aortic aneurysm in mice induced by benzo[a] pyrene

AIM: To study the role of amifostine on the formation of benzo[a]pyrene (BaP)-induced abdominal aortic aneurysm (AAA) in C57BL/6J mice and the underlying mechanism. METHODS: RAW246.7 mononuclear macrophage in vitro were divided into control group, DMSO group, BaP group, low dose (1 μmol/L) amfostine treated group, middle dose (5 μmol/L) amfostine treated group and high dose (25μmol/L) amfostine treated group. The influence of BaP on the expression of matrix metalloproteinase (MMP)-9, MMP-12, TNF-α, NF-κB in the RAW246.7 mononuclear macrophages in vitro was determined by Western blot. Male C57BL/6J mice (8 months old) were divided into control group, model group (AngII+BaP group), low dose (50 mg/kg) amfostine treated group and high dose (100 mg/kg) amfostine treated group. After 6 weeks, the abdominal aorta were isolated. The aortic tissues were subjected to HE and Masson staining. The vascular wall structure, infiltration of macrophage, the expression of MMP-9, MMP-12, TNF-α, NF-κB were evaluated by Western blot and immunochemistry staining. RESULTS: Amifostine attenuated BaP-induced expression of TNF-α, MMP-9, MMP-12, NF-κB in the RAW246.7 mononuclear macrophages (P<0.05). The results of animal experiments showed that the incidence of AAA in high dose amifostine treated group were significantly lower than that in low dose amifostine treated group and model group (P<0.05). Immunohistochemistry staining observation showed that amifostine inhibited the aortic macrophage infiltration more obviously in high amifostine treated group compared with model group and low dose amifostine treated group (P<0.05). Compared with model group and low dose amifostine treated group, the MMP-9, MMP-12, TNF-α and NF-κB expression of abdominal aorta in high amifostine treated group was reduced significantly (P<0.05). CONCLUSION: Amifostine inhibits BaP-induced activation of macrophages, and also prevents the formation of abdominal aortic aneurysm in C57BL/6J mice induced by BaP by inhibition of the NF-κB pathway, macrophage infiltration and the expression of TNF-α and MMPs.     

Effect of HMGB1 expression knockdown on invasion ability of breast cancer cells induced by TNF-α

AIM:To investigate the effect of high-mobility group box-1 (HMGB1) expression knockdown on the invasion ability of breast cancer cells induced by tumor necrosis factor-α (TNF-α). METHODS:HMGB1 siRNA was used to transfect into the breast cancer MDA-MB-231 cells. The expression of HMGB1 at mRNA and protein levels was determined by RT-qPCR and Western blot. After the MDA-MB-231 cells with HMGB1 expression knockdown were treated with TNF-α, the apoptosis rate was analyzed by flow cytometry, the cell invasion ability was measured by Transwell assay, and the cell migration ability was detected by cell scratch test. The protein expression of E-cadherin, MMP-2, N-cadherin, MMP-9 and Bax was determined by Western blot. RESULTS:The expression of HMGB1 at mRNA and protein levels in the MDA-MB-231 cells transfected with HMGB1 siRNA was significantly lower than that in the non-transfected cells (P<0.05). The apoptosis rate in the cells was increased after TNF-α treatment, and the cell invasion and migration abilities were also increased. The protein level of E-cadherin in the cells was decreased, the protein level of N-cadherin was increased, and the protein levels of MMP-2, MMP-9 and Bax were also increased (P<0.05). After the MDA-MB-231 cells with HMGB1 expression knockdown were induced by TNF-α, the apoptotic rate was increased, the invasion and migration abilities were decreased, the protein levels of E-cadherin and Bax were increased, and the protein levels of N-cadherin, MMP-2 and MMP-9 were decreased, as compared with the cells only induced by TNF-α without knockdown of HMGB1 expression (P<0.05). CONCLUSION:Knockdown of HMGB1 expression enhances the apoptosis of breast cancer cells induced by TNF-α, and inhibited the cell invasion, migration and epithelial-mesenchymal transition induced by TNF-α. The mechanism may be related with the changes of protein expression of MMP-2, MMP-9 and Bax.     

Tristetraprolin attenuates subarachnoid hemorrhage-induced early brain injury in rats

AIM: To explore the expression level of tristetraprolin (TTP) in rats after subarachnoid hemorrhage (SAH) as well as the potential role of TTP in the early brain injury (EBI) after SAH in rats.METHODS: In the first experiment setting, total 56 adult male SD rats were randomly divided into sham group and SAH group. The SAH model was performed by endovascular perforation. The brain tissues were taken out after SAH at 5 different time points (0, 12, 24, 48, 72 h and 1 week). The expression of TTP in the brain tissues was detected by Western blot. In the second experiment, a total of 60 SD rats were divided into 4 groups:sham group, SAH group, SAH+vector group and SAH+TPP group. Neurological score, brain water content and blood-brain barrier were evaluated at 48 h after SAH. TUNEL staining was performed to detect cell apoptosis in the rat brain tissue. ELISA method was used for quantitative detection of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The protein levels of TTP, Bax, Bcl-2 and cleaved caspase-3 in the rat brain tissue were detected by Western blot.RESULTS: The protein expression of TTP in the brain was downregulated markedly from 12 h after SAH, reached the lowest level at 48 h, and then had an upward trend. After modeling for 48 h, Garcia neurological score was significantly reduced, and brain water content and Evans blue (EB) content of the brain tissue of the rats in SAH group were significantly higher than those in sham group (P<0.05). SAH induced significant increases in IL-6 and TNF-α levels in the brain tissue (P<0.05). The number of TUNEL-stained cells was increased in the subcortical brain region after SAH compared with sham group. In addition, a lower level of Bcl-2 and higher levels of Bax and cleaved caspase-3 in the rat brains were observed at 48 h after SAH. However, the neurological deficit score was significantly increased, and the brain water content and EB content in the rat brains were significantly reduced in SAH+TTP group in comparison with SAH+vector group (P<0.05). Over-expression of TTP dramatically suppressed the levels of IL-6 and TNF-α in the rat brains, and reduced the number of TUNEL positive cells. Furthermore, upregulation of TTP significantly decreased the levels of cleaved caspase-3 and Bax, and evidently enhanced the expression of Bcl-2 (P<0.01).CONCLUSION: The expression of TTP is significantly decreased in early period after SAH, and enhancing the level of TTP effectively inhibits EBI following SAH in rats.     

Hydrogen sulfide inhibits inflammatory responses induced by acute myocardial ischemia in isolated rat hearts

AIM：To investigate whether hydrogen sulfide (H2S) protects the hearts against inflammatory responses induced by acute myocardial ischemia in isolated rat hearts. METHODS：Rat acute myocardial ischemia injury was induced by ligation of the left anterior descending coronary artery for 4 h, and the normal perfusate was replaced with NaHS (5 μmol/L, 10 μmol/L and 20 μmol/L) perfusate accordingly in NaHS groups 2 h after ischemia. The changes of cardiac function in the myocardial ischemic injury rats were observed. The mRNA expression of TNF-α, IL-1β, IL-6, IL-10 and ICAM-1 was detected by real-time PCR. The protein level of nuclear factor-κB (NF-κB) in the myocardial tissues was detected by Western blotting. RESULTS：The cardiac function in ischemia group was lower than that in sham group (P<0.01). Compared with ischemia group, perfusion of NaHS resulted in the improvement of the cardiac function (P<0.05 or P<0.01). Compared with sham group, the mRNA expression of TNF-α, IL-1β, IL-6 and ICAM-1 in the cardiac tissues was significantly increased, and the mRNA expression of IL-10 in the cardiac tissues was significantly decreased in ischemia group (P<0.01). Compared with ischemia group, the perfusion of NaHS significantly decreased the mRNA expression of TNF-α, IL-1β, IL-6 and ICAM-1 (P<0.05 or P<0.01). The perfusion of NaHS at concentrations of 10 μmol/L and 20 μmol/L significantly increased the mRNA expression of IL-10 (P<0.01). The protein level of NF-κB in ischemia group was markedly higher than that in sham group (P<0.01). Compared with ischemia group, the perfusion of NaHS at concentrations of 10 μmol/L and 20 μmol/L significantly decreased the expression of NF-κB (P<0.05 or P<0.01). CONCLUSION：H2S protects the hearts against acute ischemia injury through inhibition of NF-κB activation and subsequent down-regulation of NF-κB-dependent inflammatory gene expression.     

Effect of ginsenoside Rb1 on liver cell pyroptosis in hyperlipidemia rats and its possible mechanism

AIM:To discuss the mechanism of ginsenoside Rb1 against liver lipid deposition by observing the effect of ginsenoside Rb1 on liver cell pyroptosis in hyperlipidemia rats. METHODS:Totally 32 healthy SPF rats were randomly divided into control group, model group, ginsenoside Rb1 group and simvastatin group. The rats in control group was given the basic feed, while the others were given high-fat diet. The rats in ginsenoside Rb1 group and simvastatin group were given corresponding drugs. The rats in control group and model group were intraperitoneal injected with equal volume of saline. Eight weeks later, the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were tested by the automatic biochemistry analyzer. The pathological changes of the liver tissues were observed with HE staining. The protein and mRNA expression levels of pyroptosis-related factors NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were detected by Western blot and RT-qPCR. RESULTS:Compared with control group, the serum levels of TC, TG and LDL-C in model group were increased significantly (P<0.01), and the HDL-C content was decreased significantly (P<0.05). The steatotic liver cells covered the visual field. The mRNA and protein expression levels of NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were increased significantly (P<0.01). Ginsenoside Rb1 significantly decreased the serum levels of TC, TG and LDL-C (P<0.05), and significantly increased the content of HDL-C (P<0.01). Ginsenoside Rb1 also significantly decreased the degree of steatosis, and the number and size of lipid droplets. The mRNA and protein expression levels of NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were decreased significantly (P<0.05 or P<0.01). CONCLUSION:Ginsenoside Rb1 atte-nuates liver injury and inhibits liver lipid deposition in hyperlipidemia rats by reducing the expression of hepatic pyroptosis-related factors.     

Propofol inhibits LPS-induced inflammatory response in microglia via TRPV1 ion channels

AIM:To investigate the effects of propofol (P) on the inflammatory response of microglia induced by lipopolysaccharide (LPS) and the mechanisms. METHODS:Mouse microglia BV2 cells were treated with LPS at 100 μg/L to establish a neuroinflammatory injury model. The BV2 cells were divided into 4 groups:control group (C group), model group (L group), L+P group and LPS+AMG517 group (L+A group). The level of tumor necrosis factor-α (TNF-α) in the cell culture supernatant was measured by ELISA. The mRNA expression of transient receptor potential cation channel subfamily V member 1 (TRPV1) was detected by real-time PCR. The protein levels of TRPV1, TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and phosphorylated calcium/calmodulin-dependent protein kinase Ⅱ (p-CaMKⅡ) were determined by Western blot. The content of free Ca²⁺ in the microglia BV2 cells was detected by Fluo-3 AM assay. RESULTS:Compared with C group, the level of TNF-α was significantly increased in L group (P<0.01), but that in P group was not changed. Compared with L group, the level of TNF-α was significantly lower than that in L+P group within 4 h (P<0.01). Compared with C group, the mRNA expression of TRPV1 was significantly increased in L group (P<0.01). Compared with L group, the mRNA expression of TRPV1 was significantly down-regulated in L+P group (P<0.01).Compared with L group, the protein levels of TNF-α, IL-1β, IL-6 and p-CaMKⅡ and intracellular Ca²⁺ concentration were significantly lower than those in L+P group and L+A group (P<0.01). CONCLUSION:Propofol inhibits the inflammatory response of microglia by reducing the expression of TNF-α, IL-1 and IL-6, which may be related to the down-regulation of TRPV1 and p-CaMKⅡ and the reduction of intracellular Ca²⁺ concentration.