Influence of ginsenoside Re on vascular intima hyperplasia and NF-κB p65 signaling pathway in balloon-injured rats

AIM: To investigate the inhibitory effect of ginsenoside Re on intimal hyperplasia induced by balloon-injury and to explore the role of NF-κB p65 signaling pathway in the process. METHODS: SD rats(n=40) were divided into 5 groups randomly: sham operation group, model group, low-dose ginsenoside Re group, middle-dose ginsenoside Re group and high-dose ginsenoside Re group. The carotid artery intima injury model was established by 2F balloon catheters in all groups except the sham operation group. The day after modeling, the animals in model group and sham operation group were administered intragastrically with distilled water, and the rats in low-dose, middle-dose and high-dose ginsenoside Re groups were given ginsenoside Re at doses of 12.5 mg/kg, 25mg/kg and 50 mg/kg, respectively. After 14 continuous days, the morphological changes of the injured arteries were observed by HE staining and the lumen area, intima area and media area as well as the ratio of intimal area/media area were determined. The expression of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) were detected by real-time PCR. The proliferating cell nuclear antigen(PCNA) and nuclear factor-kappa B(NF-κB) p65 were examined by immunohistochemistry.RESULTS: Compared with sham operation group, the vessel cavity was narrowed(P<0.01), the mRNA levels of TNF-α and IL-1β, and the protein expression of PCNA and NF-κB p65 were increased in model group(P<0.05). Compared with model group, the vascular intimal hyperplasia was alleviated obviously(P<0.05), and the mRNA levels of TNF-α and IL-1β, and protein expression of PCNA and NF-κB p65 were decreased in medium and high-dose ginsenoside Re groups(P<0.05). CONCLUSION: Ginsenoside Re inhibits the vascular neointimal hyperplasia induced by balloon-injury in rats, and the molecular mechanism may be related to the inhibition of NF-κB p65 signaling pathway.     

Effect of IL-13 on expression of IL-1β in acute renal ischemia/ reperfusion injury in rats

AIM:To investigate the effects of IL-13 on expression of IL-1β in acute renal ischemia/reperfusion injury.METHODS:Fifty-seven male Wistar rats were randomly divided into 8 group: normal group, sham operation group, ischemia group, ischemia/reperfusion injury group(I/R), normal saline(NS)-treated group 1(C-1), NS-treated group 2(C-2), IL-13-treated group1(T-1)and IL-13-treated group 2(T-2).Rats were subjected to 45 min bilateral renal ischemia followed by reperfusion. rmIL-13 (1.5 μg/50 g body weight )was injected into the renal arteries through the abdominal aorta before ischemia(T-1) or immediately afterischemia(T-2).The serum level of IL-1β and the renal expression of IL-1β were determined in each group at 24 h post-ischemia. In addition, BUN, Cr and renal histology were also measured.RESULTS:(1)The serum level of IL-1β, gene expression and protein production of IL-1β in kidney decreased markedly in IL-13-treated groups.(2)Renal function and histology were significantly improved in IL-13-treated groups, renal injury scores decreased significantly.(3)A positive correlation were found between the serum level of IL-1β and BUN, SCr(r=0.708, P＜0.01;r=0.770, P＜0.01).CONCLUSION:These data suggest that IL-13 inhibit the expression of IL-1βand improve func-tion and histology of kidney in acute renal ischemia/reperfusion injury.     

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.     

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.     

Dexmedetomidine attenuates acute alcoholic hepatic injury in mice

AIM: To investigate the effects of dexmedetomidine (DEX) on acute alcoholic hepatic injury in mice and to explore the possible mechanisms. METHODS: Kunming mice (n=50) were randomly divided into 5 groups (n=10): normal saline control (NS) group, acute alcoholic hepatic injury model (E) group, low-dose (10 μg/kg) DEX (E+L) group, medium-dose (50 μg/kg) DEX (E+M) group and high-dose (100 μg/kg) DEX (E+H) group. The animals were sacrificed at 6 h after gavage of alcohol or normal saline. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) were measured. The livers were removed for evaluation of histological characteristics and determining the content of tumor necrosis factor-α (TNF-α) amd interleukin-1β (IL-1β) in the liver tissues by ELISA. The expression levels of cytochrome P450 2E1 (CYP2E1) and nuclear factor-κB (NF-κB) in the liver tissues were evaluated by Western blot. RESULTS: Compared with NS group, the levels of ALT, AST and TG were obviously increased in E group, which were obviously decreased in E+M and E+H groups. Compared with NS group, the levels of TNF-α, IL-1β and MDA were obviously increase in E group, which were obviously decreased in E+M and E+H groups. Compared with NS group, the activity of SOD and the content of GSH were obviously decreased in E group, which were obviously increased in E+M and E+H groups. Compared with NS group, the expression of CYP2E1 and NF-κB was obviously increase in E group, which was obviously decreased in E+M and E+H groups. Compared with NS group, ethanol induced marked liver histological injury, which was less pronounced in E+M and E+H groups. CONCLUSION: DEX has a protective effect on mouse liver with acute alcoholic injury by the involvement in the processes of antioxidation and antiinflammation, and its mechanism may be associated with the inhibition of CYP2E1 and NF-κB expression.     

Protective effect of dexmedetomidine-ulinastatin combination on lipopolysaccharide-induced acute lung injury in rats

AIM：To investigate the effects of dexmedetomidine-ulinastatin combination on acute lung injury induced by lipopolysaccharide (LPS) in rats. METHODS：Male Wistar rats were randomly divided into 5 groups: saline control group (NS group) was given saline (5 mL/kg, iv) alone; LPS group (L group) was given LPS (10 mg/kg, over 10 min); dexmedetomidine+LPS group (L+D group) was treated with the additional administration of dexmedetomidine (1 μg·kg -1·h -1) immediately after LPS injection; ulinastatin+LPS group (L+U group) was treated with the addi-tional administration of ulinastatin (50 000 U/kg, ip) immediately after LPS injection; dexmedetomidine+ulinastatin+LPS group (L+D+U group) received dexmedetomidine (1 μg·kg -1·h -1) and ulinastatin (50 000 U/kg) immediately after LPS injection. The animals were sacrificed at 6 h after LPS or NS administration. Partial pressure of arterial oxygen (PaO 2), pH and base excess (BE) were measured, and the lungs were removed for evaluation of histological characteristics and determining the concentrations of TNF-α, IL-1β, macrophage inflammatory protein 2 (MIP-2), malondialdehyde (MDA), nitric oxide (NO), prostaglandin E 2 (PGE 2) and myeloperoxidase (MPO) in lung tissues, lung wet/dry weight ratio (W/D), and albumin in brochoalveolar lavage fluid (BLAF). The pulmonary expression of nuclear factor kappa B (NF-κB) p65 was evaluated by Western blotting. RESULTS：Compared with NS group, PaO 2, pH and BE was lower in L group, which was increased by treatment with dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone. Compared with NS group, LPS induced marked lung histological injury, which was less pronounced in the animals treated with dexmedetomidine-ulinastatin combination but not dexmedetomidine or ulinastatin alone. The levels of IL-1β, IL-6, MIP-2, MDA, NO and PGE 2 in the lung tissues increased in L group compared with NS group, which were reduced by dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone. The MPO activity, MDA level and W/D increased in the lung tissues in L group compared with NS group, which was reduced by dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone. Compared with NS group, the albumin concentration in the BLAF increased, which was reduced by dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone. Compared with NS group, the expression of NF-κB p65 increased in L group, which was reduced by dexmedetomidine-ulinastatin combination but not by dexmedetomidine or ulinastatin alone.CONCLUSION：Dexmedetomidine-ulinastatin combination has a protective effect on LPS-induced acute lung injury in the rats.     

Hydrogen sulfide attenuates urosepsis-induced acute kidney injury by blocking TLR4/MyD88/PI3K signaling pathway

AIM:To explore the effect of hydrogen sulfide (H₂S) on urosepsis-induced acute kidney injury. METHODS:New Zealand white rabbits were randomly divided into control group, sham group, model (sepsis) group, NaHS treatment (NaHS) group, and NaHS combined with TAK-242 (a TLR4 inhibitor) treatment (NaHS+TAK-242) group. After treatment for 72 h, HE staining was used to measure the histopathological changes of rabbit kidney. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected by automatic biochemical analyzer. The serum levels of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), procalcitonin (PCT), interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA. The TLR4/MyD88/PI3K signaling pathway-related proteins in the kidney were determined by Western blot. RESULTS:Compared with control group, obvious damage was observed in the kidneys of septic rabbits, but the kidneys were markedly improved by treatment with NaHS. The levels of BUN, SCr, NGAL, KIM-1, PCT, IL-1β, IL-6 and TNF-α in the septic rabbits were higher than those in control group, and decreased significantly in NaHS group and NaHS+TAK-242 group. The protein levels of TLR4, MyD88, p-PI3K and p-Akt in septic rabbit kidneys were higher than those in control group. However, NaHS or NaHS+TAK-242 inhibited the activation of TLR4/MyD88/PI3K signaling pathway in the kidneys of septic rabbits. CONCLUSION:H₂S play a protective effect on the rabbits with urosepsis-induced acute kidney injury by blocking TLR4/MyD88/PI3K signaling pathway to inhibit inflammatory response.     

Effect of ischemic post-conditioning on Egr-1 and IL-1β expression in ischemia-reperfusion injured lung in rats

AIM: To investigate the protective effects of ischemic post-conditioning on the expression of early growth response factor 1 (Egr-1) and interleukin-1β(IL-1β) in ischemia-reperfusion injured lung in rats. METHODS: The model of lung ischemia-reperfusion injury was established in 24 rats and the rats were randomly allocated to 3 different groups (n=8 in each group): (1) sham group: only sham operation (thoracotomy) and no ischemia for 3 h; (2)ischemia-reperfusion group (I/R group): interruption of pulmonary perfusion and ventilation for 1 h followed by reperfusion for 2 h; (3) ischemic post-conditioning group (IPostC group): ischemic post-conditioning (5 min of reperfusion and 5 min of ischemia for 3 times) between the end of ischemia and the beginning of the reperfusion followed by reperfusion for 1.5 h. The lung tissues (prepared to small pieces of about 20 mg) were collected and homogenized at the end of the experiment. The concentration of myeloperoxidase (MPO) in the homogenate was determined. The wet to dry weight ratio (W/D) of the lung tissues was also measured at the end of reperfusion. The pathological changes of the lung tissues were observed under light microscope after reperfusion. The mRNA expression of Egr-1 and IL-1β in the lung tissues was detected by RT-PCR. RESULTS: Compared with sham group, the mRNA expression of Egr-1 and IL-1β, the levels of MPO and W/D were significantly increased in I/R group (P<0.05). The inflammatory responses of the lungs in I/R group were significantly severer than those in sham group. Compared with I/R group, the mRNA expression of Egr-1 and IL-1β, the levels of MPO and W/D in IPostC group were significantly decreased (P<0.05). The inflammatory responses of the lungs in IPostC group were also significantly attenuated. CONCLUSION: Ischemic post-conditioning significantly reduces ischemic reperfusion injury of the lung by inhibiting the expression of Egr-1 and IL-1β.     

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.     

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.     

Dexmedetomidine attenuates acute kidney injury induced by hemorrhagic shock/resuscitation in rats

AIM: To investigate the effects of dexmedetomidine on hemorrhagic shock/resuscitation (HS/R)-induced acute kidney injury (AKI) in rats, and to explore the possible mechanisms. METHODS: Wistar rats (n=32) were randomly divided into 4 groups (n=8):normal saline control group (NS group), dexmedetomidine group (D group), HS/R group and HS/R+D group. The animals were sacrificed at 6 h after resuscitation. The levels of serum creatinine (Cr) and blood urine nitrogen (BUN) were examined. The kidneys of all rats were removed for evaluation of histological characteristics, and the levels of malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and superoxide dismutase (SOD) were measured. The expression of nuclear factor-κB (NF-κB) and hemeoxygenase-1 (HO-1) was determined by Western blot. RESULTS: Compared with NS group, the levels of Cr, BUN, MDA, TNF-α and IL-1β were obviously increased in HS/R group, which were obviously decreased in HS/R+D group (P<0.05). Compared with NS group, the SOD activity was obviously decreased in HS/R group, which was obviously increased in HS/R+D group (P<0.05). Compared with NS group, the protein expression of NF-κB was obviously increased in HS/R group, which was obviously decreased in HS/R+D group (P<0.05). Compared with NS group, the protein expression of HO-1 was increased in HS/R group. Compared with HS/R group, the protein expression of HO-1 was obviously increased in HS/R+D group. Compared with NS group, HS/R induced marked kidney histological injury, which was less pronounced in HS/R+D group.CONCLUSION: Dexmedetomidine effectively protects rats against AKI caused by HS/R, and its mechanism may be associated with the increase in HO-1 expression and the inhibition of NF-κB expression.     

Role of p38MAPK in production of pro-inflammatory cytokines in Kupffer cells from severe acute pancreatitis rats

AIM:To investigate the role of p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in the Kupffer cells (KCs) production of pro-inflammatory cytokines, tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β), in severe acute pancreatitis (SAP) rats.METHODS:Sprague-Dwaley rats were randomized into three groups:①sham operation rats, ②SAP rats, ③SAP rats given the p38 MAPK inhibitor CNI-1493(10 mg/kg, iv). The SAP model was induced by the bili-pancreatic duct infusion with 5% sterile soduim taurocholate solution. Rats from each group were killed at 12 h after sham operation or SAP and Kupffer cells (KCs) were isolated. The mRNA expressions of TNF-α and IL-1β (by quantitative real-time RT-PCR) and p38 MAPK activity (by Western blot analysis) in KCs were examined. The levels of TNF-α and IL-1β in plasma were determined by ELISA.RESULTS:There was a significant acvitation of p38 MAPK in KCs harvested from SAP rats than those from sham operation rats. SAP also promoted the mRNA expressions of TNF-α and IL-1β in KCs and the plasma levels of TNF-α and IL-1β. These events were significantly inhibited by treatment with CNI-1493.CONCLUSIONS:p38 MAPK activation is one important aspect of the signaling events that may mediate the KCs production of pro-inflammatory cytokines, TNF-α and IL-1β, in SAP rats. The inhibition of the p38 MAPK may be a potential target in the prevention and treatment of SAP.     

Effects of renal denervation on inflammatory factors in a rabbit model of early atherosclerosis

AIM: To evaluate the effects of renal denervation (RDN) on the expression of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and interleukin-6 (IL-6) in a rabbit model of early atherosclerosis. METHODS: New Zealand male rabbits were divided into control group, RDN+ high-fat diet (HFD) group (RDN group), sham+HFD group (sham group) and HFD group. The rabbits in later 3 groups were fed with 2% cholesterol for 8 weeks to establish an early atherosclerosis model. The blood samples were collected to test the levels of lipids, norepinephrine (NE), TNF-α, IL-1α and IL-6. The protein expression of angiotensin Ⅱ (Ang Ⅱ) was detected by the method of immunohistochemistry. The levels of nuclear factor-κB (NF-κB) and Ang II 1 type receptor (AT1R) were evaluated by Western blot. The mRNA expression of TNF-α, IL-1α and IL-6 was determined by real-time PCR. RESULTS: After 1 d of RDN procedure, the NE level was lower in RDN group than that in sham group (P<0.01). After 8 weeks, the NE level was lower in RDN group than that in sham group and HFD group (P<0.05), and triglyceride (TG) was lower in RDN group than that in HFD group (P<0.05). The protein expression of Ang II was decreased in RDN group compared with sham group and HFD group (P<0.01). The protein expression of NF-κB was lower in RDN group than that in sham group (P<0.05). The plasma levels of TNF-α and IL-1α were reduced in RDN group compared with sham group and HFD group (P<0.05). The mRNA expression of TNF-α, IL-1α and IL-6 was reduced in RDN group compared with sham group (P<0.05). CONCLUSION: RDN inhibits sympathetic activity, decreases the plasma level of TG, and alleviates inflammatory reactions in the rabbits with atherosclerosis.     

Effect of patchouli oil on lipopolysaccharide-induced fever in rabbits

AIM:To investigate the antipyretic effect of patchouli oil on lipopolysaccharide (LPS)-induced fever in rabbits. METHODS:Male rabbits (n=42) were randomly divided into 7 groups according to their body weight and basal body temperature, including control group, model group, western medical positive group, traditional Chinese medical positive group, and high, middle and low doses (2%, 1% and 0.5%) of patchouli oil groups. Subsequently, except the controls, the rabbits were injected with LPS at a dose of 1 mL/kg (2 mg/L) through marginal ear vein to establish rabbit fever model and the rabbits in control group received the same volume of NS. The rabbits in control group and model group were injected with 0.5% Tween-80 0.5 h late, and the rabbits in the other groups were treated with correspoonding drugs. The effect of patchouli oil on the body temperature was observed, and the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the serum, and prostaglandin E2 (PGE2) and cyclic adenosine monophosphate (cAMP) in the hypothalamus were measured by radioimmunoassay. RESULTS:The body temperature and the levels of IL-1β, TNF-α, cAMP and PGE2 in model group were significant higher than those in control group. Patchouli oil notably inhibited the body temperature in the febrile rabbits. From 1.5 h to 5.5 h after administration, the body temperatures were increased by (1.06±1.55), (1.62±1.36), (1.38±1.22), (0.98±0.98) and (0.48±0.95) ℃ in high patchouli oil group, respectively. From 3.5 to 5.5 h after administration, the body temperatures were elevated by (1.47±0.73), (1.15±0.68) and (0.63±0.54) ℃ in middle patchouli oil group, respectively. A tendency of downregulation of the elevated body temperatures was observed at every time point after administration in low patchouli oil group. Patchouli oil significantly decreased the levels of TNF-α in the serum and cAMP content in the hypothalamus, and attenuated the elevated tendency of the IL-1β level in the serum and PGE2 level in the hypothalamus.CONCLUSION: Patchouli oil evidently has antipyretic effect on LPS-induced fever in the rabbits. The antipyretic mechanism might be related to the inhibition of TNF-α level in serum and cAMP content in the hypothalamus.     

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.     

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.     

Hypoxia-inducible factor-1α attenuates myocardial inflammatory injury induced by ischemia and reperfusion in rats

AIM:To investigate the role of hypoxia-inducible factor-1α (HIF-1α) stable expression in myocardial inflammatory injury induced by ischemia and reperfusion (I/R) in rats. METHODS:Male Wistar rats were randomly divided into 4 groups:sham operation (sham) group, I/R group, HIF-1α stabilizer dimethyloxalyl glycine (DMOG)+I/R group and HIF-1α inhibitor YC-1+I/R group. The protein expression of myocardial Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) was determined by Western blot. The mRNA levels of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, TLR4 and NF-κB were detected by real-time PCR. The myeloperoxidase (MPO) activity in the myocardial tissues was measured. HE staining was used to observe the infiltration of inflammatory cells. RESULTS:HIF-1α decreased the infiltration of inflammatory cells, the MPO activity, and the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the myocardial tissues. HIF-1α also reduced the expression of TLR4 and NF-κB at mRNA and protein levels (P<0.05). CONCLUSION:The stable expression of HIF-1α has an anti-inflammatory effect on the myocardial tissues after I/R injury in rats. The mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway.     

Protective effect of all-trans retinoic acid pretreatment against rat intestinal injury induced by hepatic inflow occlusion

AIM：To investigate the effect of all-trans retinoic acid (ATRA) on the intestinal injury induced by hepatic inflow occlusion (HIO) and its mechanisms. METHODS：Thirty-two male Sprague-Dawley rats were randomly divided into four groups: sham group, HIO group, dimethyl sulfoxide (DMSO) + HIO group and ATRA (15 mg·kg-1·d-1) + HIO group. The hepatoduodenal ligament of the rats in the latter three groups was occluded (Pringle manoeuvre) by clamp for 30 min. After reperfusion for 2 h by release of the clamp, samples of distal ileum and serum were collected. Histological changes and Chiu’s scores of the ileac mucosa were evaluated under light microscope. Serum content of diamine oxidase (DAO), and ileac tissue levels of malonaldehyde (MDA) and superoxide dismutase (SOD) were analyzed by colorimetry. Serum concentrations of interleukin (IL)-1β and tumor necrosis factor (TNF)-α were evaluated by enzyme-linked immunosorbent assay method. Expression of manganese superoxide dismutase (MnSOD) in cytoplasm and nuclear factor kappa B (NF-κB) p65 in nucleus was assessed by Western blotting. RESULTS：Compared with sham group and DMSO+HIO group, ATRA significantly reduced the mucosal Chiu’s scores, the serum content of DAO and the tissue level of MDA, enhanced the serum activity of SOD and the protein expression of MnSOD, and decreased the content of NF-κB p65 in nucleus (all P<0.05). Subsequently, ATRA significantly reduced the levels of TNF-α and IL-1β in serum (P<0.05). CONCLUSION：ATRA can attenuate rat intestinal injury induced by HIO through improving the antioxidant capacity of tissue, inhibiting the activation of NF-κB and suppressing the overexpression of pro-inflammatory factors.     

Lactulose preconditioning protects against intestinal ischemia-reperfusion injury in rats

AIM: To investigate the protective effect of lactulose preconditioning on intestinal ischemia-reperfusion (IR) injury in rats. METHODS: Thirty Sprague-Dawley rats were randomly divided into 3 groups: sham operation group, IR group and IR plus lactulose preconditioning group. Lactulose was intragastrically administered in lactulose group 7 days prior to operation, and the equal volume of saline was administered in the other 2 groups. The intestinal IR injury was induced in IR group and IR+lactulose group using bulldog clamps on superior mesenteric artery by 30 min of ischemia followed by 60 min of reperfusion. Following reperfusion, the serum samples were collected for estimating the levels of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and IL-1β. Segments of terminal jejunum were rapidly fixed in 4% paraformaldehyde, and HE staining was applied to assess the histopathology. Apoptosis in intestinal epithelium was determined by the technique of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The samples of terminal jejunum were also taken for measuring malondialdehyde,superoxide dismutase and the expression of cleaved caspase-3. RESULTS: Lactulose preconditioning significantly attenuated the severity of intestinal IR injury, with inhibition of IR-induced apoptosis. Moreover, lactulose preconditioning significantly limited the release of cytokines and lipid oxidation. CONCLUSION: Lactulose preconditioning has a protective effect on intestinal ischemia reperfusion by inhibiting IR-induced apoptosis and oxidative stress.