Inflammatory response in rat kidney with contrast-induced nephropathy

AIM：To observe the expression of tumor necrosis factor α (TNF-α) and nuclear factor κB (NF-κB) in the renal tissue of the rats with contrast-induced nephropathy (CIN). METHODS：Male Sprague-Dawley rats (n=96) were randomly divided into control group (n=48) and CIN group (n=48). The model rats in CIN group were intravenously injected with iodinated contrast media (76% compound diatrizoate injection,10 mL/kg), while the rats in control group were injected with the same volume of saline. Six rats in each group were sacrificed at 6 h, 12 h, 24 h, 48 h, 72 h, 5 d, 10 d and 15 d after intravenous injection, respectively, and the blood and kidney samples of the rats were obtained. The renal tubular injury was assessed by histological examination (HE staining). The expression of kidney injury molecule-1 (KIM-1), TNF-α and NF-κB at mRNA and protein levels in the renal tissues were semiquantitatively measured by the methods of RT-PCR and immunohistochemistry, respectively. The correlations between the expression of TNF-α, NF-κB and tubular injury score, KIM-1 expression in renal tissue of CIN group were analyzed. RESULTS：The levels of serum creatinine (SCr) and blood urea nitrogen (BUN) in control group were not changed between different time points (P>0.05). The levels of SCr and BUN in CIN group displayed significant increases at different time points (except 15 d) compared with control group (P<0.05). The renal tubular injury score in CIN group was significantly higher at all time points than that in control group (P<0.05). The expression of KIM-1, TNF-α and NF-κB at mRNA and protein levels up-regulated significantly at 6 h and the peaking of KIM-1 expression was at 24 h, while the peaking of TNF-α and NF-κB expression was at 48 h in CIN group. The expression of KIM-1,TNF-α and NF-κB was significantly increased in CIN group compared with control group except at 15 d (P<0.05). The expression of TNF-α and NF-κB at mRNA and protein levels showed close correlations with renal tubular injury score (r=0.843, 0.758, 0.743 and 0.707, P<0.05). The expression of TNF-α and NF-κB at mRNA and protein levels was also positively correlated with KIM-1 expression (r=0.863, 0.807, 0.839 and 0.855, P<0.05). CONCLUSION：The expression of TNF-α and NF-κB at mRNA and protein levels in the renal tissues of CIN group is up-regulated and is closely related with renal tubular injury, indicating that the inflammatory response is involved in the pathogenesis of CIN.     

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.     

Effects of tripterygium glycosides combined with dexamethasone on TLRs/NF-κB signaling pathway in EAE rats

AIM: To investigate the role of TLRs/NF-κB pathway in experimental allergic encephalomyelitis (EAE) rats treated with tripterygium glycosides (TG) + dexamethasone (DX). METHODS: Lewis rats were used in the study and divided into control group, EAE model group, therapy 1 group (EAE rats treated with DX) and therapy 2 group (EAE rats treated with DX+TG). The mean clinical score of the rats was determined. The expression of TLR4 and TLR9 at mRNA and protein levels was detected by the methods of real-time quantitative RT-PCR and immunohistochemistry. The protein level of NF-κB p65 was also measured. The levels of TNF-α, IL-1β and IL-6 were assayed by ELISA. RESULTS: The mean clinical scores at 5th, 16th and 20th day were lower in therapy 1 group and therapy 2 group than that in EAE model group. The mean clinical score in therapy 2 group was even lower than that in therapy 1 group. At the 16th day (the peaking period), the mRNA expression of TLR4 and TLR9 in therapy 1 group and therapy 2 group were obviously lower than that in EAE model group. The protein levels of TLR4, TLR9 and NF-κB p65 were also significantly lower in therapy 1 group and therapy 2 group than those in EAE model group at peak stage of EAE. The levels of TNF-α, IL-1β and IL-6 were lower in therapy1 group and therapy2 group than those in EAE model group. The significant differences of the mean clinical score, the mRNA expression of TLR4 and TLR9, the positive ratio of NF-κB p65 and the levels of TNF-α, IL-1β and IL-6 between therapy 1 group and therapy 2 group were found. The result of orthogonal factorial analysis of variance indicated that the difference of therapeutic effect between DX and DX+TG was significant (F=75.749, P<0.01). CONCLUSION: The TLRs/NF-κB pathway takes part in the pathological process of EAE. TG combined with DX alleviates the symptoms of EAE by suppressing inflammatory and immunological reactions of EAE.     

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.     

Effects of icariin preconditioning on inflammatory responses in myocar-dial ischemia-reperfusion injury

AIM:To observe the effects of icariin on myocardial ischemia-reperfusion injury. METHODS:The left anterior descending coronary artery was ligated for 30 min and then loosened for 2 h to establish the rat model of myocardial ischemia-reperfusion injury. Forty-eight healthy adult male SD rats weighing 250~300 g were randomly divided into sham group, model group, low-, middle-and high-dose icariin groups, and aspirin group. The morphological changes of the myocardium were observed by HE staining. The protein expression of NF-κB p65 in the myocardial nucleus was determined by the method of immunohistochemistry. The content of tumor necrosis factor α (TNF-α) in the myocardial tissues was detected by Western blotting. The level of interleukin 1β (IL-1β) in the serum was measured by ELISA. The activity of myeloperoxidase (MPO) in the myocardial tissues was assayed by colorimetry. RESULTS:Compared with sham group, TNF-α content, IL-1β concentration, NF-κB expression and MPO activity in all other groups increased. Compared with model group, TNF-α content, IL-1β concentration, NF-κB expression and MPO activity in low-, middle- and high-dose icariin groups and aspirin group all decreased. No significant difference of the above parameters between high-dose icariin group and aspirin group was observed. CONCLUSION: Icariin preconditioning reduces inflammatory responses in the process of myocardial ischemia-reperfusion injury in a dose-dependent manner.     

Effect of peroxisome proliferator-activated receptor γ agonist rosiglitazone on intestinal injury in rats undergoing orthotopic autologous liver transplantation by inhibiting inflammatory response

AIM: To investigate the effect of rosiglitazone, a peroxisome proliferators-activated receptor γ(PPARγ) agonist, on the expression of PPARγ, the activation of NF-κB and intestine injury in the rats undergoing orthotopic autologous liver transplantation(OALT).METHODS: Sprague-Dawley male rats were randomly divided into 4 groups:control group, sham group, OALT group and rosiglitazone(0.3 mg/kg, iv) pretreatment(ROS+OALT) group. The OALT model was established, and the intestinal tissues were collected 8 h after the liver reperfusion. The intestinal tissue sections were stained to visualize the damage. The expression of PPARγ and NF-κB in the tissues, the concentrations of diamine oxidase(DAO) and fatty acid-binding protein 2(FABP2) in the serum and the concentration of TNF-α and IL-6 in the tissues were measured.RESULTS: Compared with sham group, the intestinal mucosa of the rats showed obvious pathological injury after liver reperfusion in OALT group and ROS group, the Chiu,s scores of intestinal mucosa was significantly higher, and the serum concentrations of DAO and FABP2 increased(P<0.05). After rosiglitazone pretreatment, the injury of intestinal mucosa of the rats was alleviated, the Chiu,s scores was lower and the serum concentrations of DAO and FABP2 decreased(P<0.05), the PPARγ expression was obviously up-regulated in the intestinal tissues, the nuclear translocation of NF-κB was reduced and the concentrations of IL-6 and TNF-α were decreased.CONCLUSION: During perioperative period of OALT in rats, the inflammatory responses are obvious. Furthermore, obvious intestinal injury occurs. PPARγ agonist rosiglitazone obviously up-regulates PPARγ expression and inhibits the inflammation in the intestines, thus protecting against intestinal injury in rats undergoing OALT.     

Effects of Huaiyu decoctum on serum concentrations of TNF-α, IL-6 and IL-10 in rats after anorectal operation

ATM: To investigate the effects of Huaiyu decoctum on the serum concentrations of TNF-α, IL-6 and IL-10 in rats after anorectal operation. METHODS: Sprague-Dawley rats (n=40) were randomly divided into 4 groups:normal group, model group, low-dose Huaiyu decoctum group and high-dose Huaiyu decoctum group. The concentrations of TNF-α, IL-6 and IL-10 in the rat serum were measured by ELISA. The pathologic changes of the anorectal tissues were observed under microscope with HE staining. The protein expression of ICAM-1, VCAM-1 and NF-κB was determined by Western blotting. RESULTS: After Huaiyu decoctum administration, TNF-α and IL-6 concentrations in the serum were significantly decreased, and IL-10 concentration was increased as compared with model group. Moreover, Huaiyu decoctum markedly attenuated edema and hyperemia in the rats after anorectal operation. The protein expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and NF-κB in the anorectal tissues was obviously inhibited by Huaiyu decoctum treatment. CONCLUSION: Huaiyu decoctum improves the recovery of anorectal tissues after operation by decreasing the serum concentrations of TNF-α, IL-6 and IL-10, and reducing the protein expression of ICAM-1, VCAM-1 and NF-κB in the anorectal tissues.     

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 TRPV1 activation on lipopolysaccharide-induced lung inflammatory injury in mice

AIM:To investigate the effects of transient receptor potential cation channel subfamily V member 1 (TRPV1) activation by capsaicin on the inflammation and its underlying mechanisms in lipopolysaccharide (LPS)-induced lung injury in mice. METHODS:A total of 108 specific pathogen-free male ICR mice were randomly divided into 6 groups: normal control group, capsaicin (CAP) control group, capsazepine (CAPZ) control group, endotoxemia group, CAP treatment group and CAPZ treatment group. LPS was intraperitoneally injected 30 min after the subcutaneous injection of CAP or CAPZ. After modeling, the levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), IL-10, substance P (SP) and calcitonin gene-related peptide (CGRP) in the lung were measured by ELISA. The expression of Toll-like receptor 4 (TLR4) and nuclear factor κB (NF-κB) in the lung tissue was assessed by Western blotting. The pathological changes of the lung tissue were observed under light microscope. RESULTS:The expression of TNF-α, IL-6, IL-10 and NF-κB in the lung tissues at 3 h, 8 h and 16 h was dramatically higher in endotoxemia group than that in normal control group. Compared with endotoxemia group, the levels of TNF-α, IL-6 and nuclear NF-κB in CAP treatment group at 3 h, 8 h and 16 h were obviously decreased, but the level of IL-10 was increased. The changes of the factors mentioned above in CAPZ treatment group were absolutely adverse to those in CAP treatment group. The levels of SP and CGRP were significantly higher in endotoxemia group and CAP control group than those in normal control group, but those in CAPZ control group were lower. Compared with endotoxemia group, SP and CGRP were markedly increased in CAP treatment group and were obviously decreased in CAPZ treatment group. The level of TLR4 in endotoxemia group was distinctly higher than that in normal control group at 3 h, 8 h and 16 h. However, as compared with endotoxemia group, the expression of TLR4 in CAP treatment group and CAPZ treatment group didn’t change much. At 8 h and 16 h after modeling, the degree of lung damage was also decreased in CAP treatment group as compared with endotoxemia group, while that in CAPZ treatment group was aggravated. CONCLUSION: TRPV1 activation obviously inhibits the increase in TNF-α, IL-6 and NF-κB in the lung tissue of endotoxemia mice, and promotes the increase in the anti-inflammatory factor IL-10, as well as the levels of SP and CGRP, but has no effect on the expression of TLR4.     

Effect of Yiqi Huayu Huatan decoction on expression of KLF15, HMGB1, NF-κB and its downstream inflammatory factors in rats with UUO-induced renal interstitial fibrosis

AIM: To observe the effect of Yiqi Huayu Huatan decoction (YHHD) on unilaterral ureteral obstruction (UUO)-induced renal interstitial fibrosis in rats, and to investigate the possible mechanism. METHODS: Female SD rats (n=48) were randomly divided into sham group, model group, telmisartan group, and low-, middle-and high-dose YHHD groups, with 8 rats in each group. The UUO model rats was established by ligating left ureter. The rats in sham group and model group were treated with equal volume of normal saline, others were treated with the corresponding drugs daily. After 12 weeks, the rats were sacrificed. The serum samples were collected for determining the concentrations of cystatin C (Cys-C) and uric acid (UA). The morphological changes of the renal tissue were observed by PAS staining. The collagen fiber was observed by Masson staining. The mRNA expression of Krüppel-like factor 15 (KLF15), high-mo-bility group box protein 1 (HMGB1), nuclear factor-κB (NF-κB), IκB, monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), fibronectin (FN), collagen type I (Col I) and Col-Ⅳ was detected by real-time PCR. The protein expression of KLF15, HMGB1 and NF-κB was detected by Western blot. The protein expression of MCP-1 was determined by the method of immunohistochemistry. RESULTS: Compared with sham group, the deposition rate of collagen fibers and the concentration of Cys-C in model group were significantly increased (P<0.05), the mRNA and protein expression of KLF15 was significantly down-regulated (P<0.05), while the mRNA expression of HMGB1, NF-κB, IκB, MCP-1, IL-1β, TNF-α, FN, Col I and Col Ⅳ and the protein expression of HMGB1, NF-κB and MCP-1 were significantly up-regulated (P<0.05). Compared with model group, the deposition rates of collagen fibers in middle-and high-dose YHHD groups and telmisartan group were significantly decreased (P<0.05), with down-regulated protein expression of HMGB1 and NF-κB and mRNA expression of IL-1β and TNF-α (P<0.05). The protein expression of KLF15 was significantly up-regulated in high-dose YHHD group and telmisartan group (P<0.05), while the protein expression of MCP-1 and the mRNA expression of FN were significantly down-regulated (P<0.05). The mRNA expression of KLF15 was significantly up-regulated in high-dose YHHD group (P<0.05), while the mRNA expression of MCP-1, Col I and Col IV was significantly down-regulated (P<0.05). The mRNA expression of NF-κB and IκB was significantly down-regulated and the concentration of Cys-C was significantly decreased in each dose of YHHD groups and telmisartan group (P<0.05). No significant difference of UA level among the groups was observed. CONCLUSION: YHHD alleviates renal interstitial fibrosis in a dose-dependent manner, and YHHD at high dose shows the most obvious effect. The mechanism may be associated with the up-regulation of KLF15 and the down-regulation of HMGB1, NF-κB and its downstream inflammation-related factors in the renal tissue.     

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.     

Inhibitory effects of right cervical sympathetic trunk transection on inflammatory response after acute myocardial infarction in rats and its influence on HMGB1/TLR4/NF-κB signaling pathway

AIM: To observe the effects of transection of right cervical sympathetic trunk (TCST) on inflammatory response and expression of high mobility group box 1 (HMGB1) and TLR4/NF-κB signaling pathway in the rats after acute myocardial infarction (AMI). METHODS: AMI model was established by ligation of left anterior descending coronary artery in SD rats, then the model rats were randomly divided into MI group and MI+TCST group. MI+TCST model was performed by transection of right cervical sympathetic trunk after left anterior descending coronary artery ligation. The rats in MI group and MI+TCST group were divided into 1, 3, 7, 14 and 28 d subgroups, and another sham operation group threading without ligation, with 8 rats in above each group. After modeling for 4 weeks, the cardiac function was measured by echocardiography. All rats were killed to harvest the hearts for mesuring cardiac hypertrophy index. The myocardial tissue close to infarction was observed with HE staining. The relative mRNA expression levels of HMGB1, tumor necrosis factor α(TNF-α) and interleukin (IL)-6 at different time points were detected by real-time PCR. The protein expression of HMGB1 and TLR4 at different time points after AMI was determined by Western blot. The effect of transection of right cervical sympathetic trunk on the expressions of HMGB1 and TLR4/NF-κB signaling pathway was also analyzed.RESULTS: Compared with the MI group, left ventricular ejection fraction (LVEF) and left ventricular shorterning fraction (LVFS) were significantly higher (P<0.05), left ventricular end-diastole dimension (LVEDd), left ventricular end-systole dimension (LVESd) and cardiac hypertrophy index were significantly lower (P<0.05), and the mRNA levels of HMGB1, TNF-α and IL-6 decreased significantly in MI+TCST group (P<0.05). Western blot results revealed that the protein expression level of HMGB1 increased in the infarct border zone at 3 d, and reached its peak at 7 d, then gradually decreased, and at 28 d after MI in MI group was still significantly higher than that in sham group (P<0.05). The protein expression of TLR4 was consistent with that of HMGB1. Transection of right cervical sympathetic trunk reduced protein expression of HMGB1 and TLR4/NF-κB signaling pathway-related proteins (P<0.05).CONCLUSION: Transection of right cervical sympathetic trunk improves ventricular remodeling and maintaining cardiac function. The mechanism may be related to inhibiting HMGB1/TLR4/NF-κB signaling pathway to reduce inflammatory response.     

Artemisinin attenuates intestinal epithelial barrier damage induced by LPS

AIM: To investigate the effect of artemisinin on lipopolysaccharide(LPS)-induced intestinal epithelial barrier damage in IEC-6 cells and its molecular mechanism. METHODS: Cultured IEC-6 cells were divided to 5 groups: control group, LPS(100 mg/L) group and LPS+Artemisinin(30, 50 and 100 μmol/L) groups. The cytotoxicity was detected by MTT assay. The releases of TNF-α, IL-1β and IL-6 in the IEC-6 cells were measured by ELISA. The transepithelial electrical resistance(TER) was detected by electrical resistance tester, and the horseradish peroxidase(HRP) flux permeability were analyzed by a microplate reader. The expression of tight junction proteins, ZO-1, claudin-1 and occludin, and the expression of TLR4/MyD88/NF-κB at mRNA and protein levels were determined by RT-qPCR and Western blot. RESULTS: Artemisinin alone(up to 100 μmol/L) or in combination with LPS(100 mg/L) was not toxic to IEC-6 cells. Compared with control group, the releases of TNF-α, IL-1β and IL-6 in the culture supernatant of IEC-6 cells significantly increased after treatment with LPS. The expression of TLR4/MyD88/NF-κB was activated by LPS. LPS down-regulated the protein expression of ZO-1, claudin-1 and occludin. However, artemisinin treatment decreased the releases of TNF-α, IL-1β and IL-6 in the culture supernatant of IEC-6 cells. The expression of TLR4/MyD88/NF-κB at mRNA and protein levels was gradually reduced after treatment with artemisinin. In addition, artemisinin upregulated the protein expression of ZO-1, claudin-1 and occludin significantly(P<0.01) in a dose-dependent manner. CONCLUSION: Artemisinin attenuates LPS-induced intestinal epithelial barrier damage by inhibiting TLR4/MyD88/NF-κB activation in the IEC-6 cells.     

Inhibitory effect of madecassoside on LPS-stimulated microglia

AIM: To observe the inhibitory effect of madecassoside on the LPS-stimulated microglia and to investigate its possible mechanism. METHODS: Microglia cells of neonatal Sprague-Dawley (SD) rats were cultured, isolated and purified. Microglia cells were activated with lipopolysaccharide (LPS). The inhibitory effect of madecassoside on microglia was measured by MTT assay. Tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β) were detected by ELISA. Cell cycle and apoptotic rate were evaluated by flow cytometry. The expression of TLR4 was detected by Western blotting. The expression of NF-κB was detected by RT-PCR. RESULTS: LPS induced the proliferation of microglia and release inflammatory cytokines significantly. Compared with LPS group, madecassoside inhibited the proliferation of microglia induced by LPS in a dose dependent manner. The IC₅₀ value of madecassoside was 10.97 nmol/L to microglia after incubation for 48 h. Madecassoside also decreased the levels of TNF-α and IL-6, increased the ratios of microglia at the G₂ phase and the apoptotic rate, decreased the expression of TLR4 and NF-κB significantly (P<0.05). CONCLUSION: Madecassoside has inhibitory effects on the proliferation of LPS-stimulated microglia, by which the mechanism may be related to inhibition of the expression of TLR4 and NF-κB, change of cell cycle distribution and induction of microglia apoptosis.     

Effects of angiopoietin 4 on lipopolysaccharide-induced injury of human umbilical vein endothelial cells

AIM:To observe the effects of angiopoietin 4 (Ang-4) on lipopolysaccharide (LPS)-induced injury of human umbilical vein endothelial cells (HUVECs). METHODS:The EnVision immunohistochemical method was used to identify the HUVECs. After pre-treated with different doses of Ang-4 for 0.5 h, HUVECs was exposed to LPS at concentration of 10 mg/L for 24 h. The cell viability was evaluated by MTT assay. The content of tumor necrosis factor-alpha (TNF-α) in the supernatant and the concentrations of intracellular and supernatant von Willebrand factor (vWF) were detected by ELISA. The mRNA levels of Toll-like receptor 4 (TLR4), NF-κB p65 and TNF-α were determined by real-time PCR. RESULTS:Factor Ⅷ in the cytoplasm was positive in the HUVECs.Compared with normal group, LPS reduced the cell viability (P<0.01), and significantly increased the secretion of TNF-α and vWF (P<0.01). The mRNA expression of TLR4, NF-κB p65 and TNF-α also increased (P<0.01). Ang-4 at concentration of 100 μg/L enhanced the cell viability (P<0.01), reduced the content of vWF and TNF-α, and inhibited the LPS-induced increases in the mRNA levels of TLR4, NF-κB p65 and TNF-α (P<0.01). CONCLUSION: Ang-4 antagonizes LPS-induced damage in HUVECs by inhibiting TLR4-NF-κB p65-TNF-α signaling pathways.     

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.     

Protective effects of taurine on LPS-induced myocardial damage in rats

AIM: To investigate the effects of taurine on lipopolysaccharide (LPS)-induced myocardial damage in rats. METHODS: Healthy male SD rats (n=30) were randomly divided into control group (CON), LPS model group (LPS) and taurine treatment group (TAU). The rats in CON group and LPS group were intravenously injected with normal saline, and the rats in TAU group were injected with taurine (100 mg/kg). After 2 h, the rats in LPS group and TAU group were intraperitoneally injected with LPS at 10 mg/kg, and the rats in CON group were injected with normal saline. Six hours after injection of LPS, the blood samples were collected for determination of superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) levels. The myocardial tissues were processed for histological examination and the analysis of Western blot. RESULTS: Compared with CON group, LPS significantly reduced SOD activity in the serum and heme oxygenase 1 (HO-1) protein expression in the myocardial tissues, increased the serum content of MDA and levels of TNF-α and IL-6. LPS also significantly elevated the levels of TNF-α and IL-6, and up-regulated the cyclooxygenase-2 (COX-2) expression and phosphorylation of nuclear factor kappa B (NF-κB) in the myocardial tissues. Taurine pretreatment significantly elevated SOD activity and HO-1 protein expression level, decreased the levels of COX-2, TNF-α, IL-6 and phosphorylated NF-κB. Histological observation showed that taurine reduced inflammatory response in the myocardial tissue. CONCLUSION: Taurine attenuates LPS-induced myocardial damage in rats. The beneficial effects of taurine may be associated with its reduction of p-NF-κB/COX-2 signaling by activation of HO-1/CO.     

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 recombinant rat CC16 protein on LPS-induced expression of TNF-α, IL-6 and IL-8 in rat tracheal epithelial cells

AIM: To explore the effect of recombinamt rat CC16 protein (rCC16) on LPS-induced expression of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and IL-8 in the rat tracheal epithelial (RTE) cells.METHODS: The RTE cells were incubated with rCC16 at concentrations of 0.5, 1.0 and 2.0 mg/L in serum-free media for 2 h prior to LPS (0.1 mg/L) treatment for further 24 h. The cells were harvested for assessing the mRNA levels of TNF-α, IL-6 and IL-8 by RT-qPCR. The cell culture supernatants were collected for analyzing the protein levels of TNF-α, IL-6 and IL-8 by ELISA. In addition, the nuclear translocation of nuclear factor-κB (NF-κB) p65 was tested by Western blot.RESULTS: rCC16 inhibited LPS-induced IL-6 and IL-8 expression at both mRNA and protein levels in the RTE cells in a concentration-dependent (0~2 mg/L) manner, as demonstrated by RT-qPCR and ELISA. However, no concentration-dependent manner between the dose of rCC16 and TNF-α expression was observed, and rCC16 inhibited LPS-induced TNF-α expression at lower concentration (0.5 mg/L). rCC16 concentration-dependently inhibited the effects of LPS on the level of nuclear translocation of NF-κB p65.CONCLUSION: rCC16 suppresses LPS-mediated TNF-α, IL-6 and IL-8 production through inactivation of NF-κB activity in RTE cells.[KEY WORDS] CC16 protein; Airway inflammation; LPS; Inflammatory mediators; Nuclear factor-κB