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.     

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.     

Effect of Notch1/Hes1 signaling pathway on proliferation and differentiation of AECⅡ by regulating expression of C/EBPα

AIM: To investigate whether Notch1/Hes1 signaling pathway regulates the expression of CCAAT/enhancer binding protein alpha (C/EBPα), thus affecting the proliferation and differentiation of type Ⅱ alveolar epithelial cells (AECⅡ). METHODS: Human AECⅡ were cultured in vitro, and randomly divided into control group, activator group (adding Notch pathway activator Jagged1 protein, 500 μg/L) and inhibitor group (adding Notch inhibitor DAPT, 10 μmol/L). AECⅡ in each group were collected after 24 h of intervention. The expression of Notch1, Hes1 and C/EBPα at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell proliferation ability of the AECⅡ was measured by living cell counting and CCK-8 assay. The cell cycle distribution and differentiation of the AECⅡ were analyzed by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression levels of Notch1, Hes1 and C/EBPα were significantly increased in activator group (P<0.05), AECⅡ entered G₂/M phase from S phase, the proliferation of AECⅡ was increased, and the differentiation of AECⅡ was reduced (P<0.05). The mRNA and protein expression levels of Notch1, Hes1 and C/EBPα were significantly reduced in inhibitor group (P<0.05), the cell cycle of AECⅡ cells was arrested in G₀/G₁ phase, the proliferation of AECⅡ cells was reduced, and the differentiation of AECⅡ cells was increased (P<0.05). CONCLUSION: Notch1/Hes1 signaling pathway regulates the expression of C/EBPα and affects the proliferation and differentiation of AECⅡ.     

Effect of astragaloside IV on neurological function in acute cerebral he-morrhage rats

AIM: To investigate the effect of astragaloside IV on neurological function and the protein expression of neuron-specific enolase (NSE), Notch1 and NF-κB in acute cerebral hemorrhage (ACH) rats.METHODS: ACH model in rats was established via injection of autologous blood, and the rats were divided into 4 groups:sham, ACH, ACH+astragaloside IV (100 mg/kg) and ACH+astragaloside IV (200 mg/kg) groups. The impairment of neurological function in each group was graded, and the brain coefficient and water content were calculated. The serum level of NSE was measured by ELISA. Additionally, the expression of Notch1 and NF-κB was determined by Western blot.RESULTS: Astragaloside IV improved the neurological function and decreased the brain coefficient and water content in ACH rats. Moreover, the ACH-induced increase in NSE was inhibited after astragaloside IV treatment. Similarly, astragaloside IV also significantly attenuated the expression of Notch1 and NF-κB in ACH rats.CONCLUSION: Astragaloside IV attenuates the impairment of neurological function in ACH rats, which may be through decreasing the NSE level and down-regulating the expression of Notch1 and NF-κB.     

Inhibitory effect of recombinant human endostatin on angiogenesis in atherosclerotic plaque of rats by regulating Dll4/Notch pathway

AIM: To observe the inhibitory effect of recombinant human endostatin (rhES) on plaque angiogenesis, and to explore the regulatory mechanism of Dll4/Notch pathway in the anti-angiogenic effect of rhES. METHODS: Male Wistar rats were randomized into 3 groups:normal control group (N group), atherosclerotic model group (AS group), and rhES treated group (AS+rhES group). The rats in N group were fed a normal diet, while the remaining 2 groups were established to atherosclerotic rat model via high-cholesterol diet, intraperitoneal injection of vitamin D₃ and aortic balloon injury. The rats in AS+rhES group received intraperitoneal injection of rhES. The blood total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), C-reactive protein (CRP), interleukin-1 (IL-1) and troponin I (TnI) were measured. The atherosclerotic abdominal aortas were taken for pathological observation. Immunohistochemical staining was used to measure the density of neovessels in the plaques, which were marked by CD31. The protein levels of Dll4 and Notch1 in the aortas were analyzed by Western blot. RESULTS: The levels of blood TC, TG, LDL-C, CRP and IL-1 in AS group and AS+rhES group were much higher than those in N group (P<0.05), and no statistical difference between AS group and AS+rhES group was observed. The expression of CD31 in AS group was the highest among all groups. Compared with AS group, the density of neovessels in the plaques of AS+rhES group decreased significantly (P<0.05). The protein expression of Dll4 and Notch1 in AS group was lower than that in N group (P<0.05). Compared with AS group, the protein expression of Dll4 and Notch1 increased significantly (P<0.05). CONCLUSION: rhES has the ability to inhibit plaque angiogenesis in rats. The activation of Dll4/Notch pathway may be the mechanism of rhES in inhibiting plaque angiogenesis.     

Effects of Jiedu-Qingfei mixture on NF-κB and p38 MAPK pathways in lung tissues of rats with Mycoplasma pneumoniae infection

AIM: To observe the therapeutic effect of Jiedu-Qingfei mixture on Mycoplasma pneumoniae (MP)-infected rat lung tissues and to explore its mechanism. METHODS: SD rats (n=40) were randomly divided into 4 groups:blank control group, model group, Jiedu-Qingfei group and positive control group, with 10 rats in each group. The rats in experimental groups were slowly dripped with 1×10⁹ CFU/L MP solution into their nostrils for 4 d. One rat in each group was sacrificed for MP nucleic acid detection at the second day after inoculation, and the other rats were given gavage therapy. The rats in blank control group and model group were intragastrically given the same volume of normal saline, the rats in Jiedu-Qingfei group were given 8 mL/kg Jiedu-Qingfei mixture daily for 4 weeks, and the rats in psoitive control group were given dexmethasone sodium phosphate (0.5 mg·kg^-1·d^-1). After the experiment, the rats were killed. The serum and bronchoalveolar lavage fluid (BALF) were collected for detecting the levels of interleukin-12 (IL-12), IL-13 and TNF-α by ELISA. The right lung tissues were used for pathological observation and HE staining, while the left lung tissues were used to detect the expression of NF-κB p50, I-κBα and p38 mitogen-activated protein kinase (p38 MAPK) at mRNA and protein levels. RESULTS: The results of MP nucleic acid detection showed that all the rats except blank control group were MP nucleic acid positive, indicating that the rat model of MP infection was successfully established. On the 1st day of the treatment, the pathological scores of the lung tissues in model group and Jiedu-Qingfei group were significantly higher than those in blank control group (P<0.05). After treatment, the pathological scores of the lung tissues in mo-del group were significantly higher than those in blank control group and Jiedu-Qingfei group. The levels of IL-12 in the serum and BALF in model group were significantly lower than those in blank control group after MP infection (P<0.05), while those after treatment with Jiedu-Qingfei mixture were significantly higher than those in model group (P<0.05). The levels of IL-13 and TNF-α in the serum and BALF of MP-infected rats were increased significantly, while those after treatment with Jiedu-Qingfei mixture were significantly lower than those in model group (P<0.05). The mRNA expression levels of NF-κB p50 and p38 MAPK in model group were increased significantly (P<0.01). After treatment, the mRNA expression levels of NF-κB p50 and p38 MAPK were decreased significantly compared with model group (P<0.01). The mRNA expression level of I-κBα in model group was significantly lower than that in control group. After treatment, the mRNA expression of I-κBα in Jiedu-Qingfei group was significantly higher than that in model group (P<0.05). The protein levels of NF-κB p50 and p38 MAPK in the lung tissues of model group were significantly higher than those of blank control group. After treatment, the protein expression of NF-κB p50 and p38 MAPK was decreased significantly. The protein level of I-κBα in model group was significantly lower than that in blank control group, and after treatment with Jiedu-Qingfei mixture, the protein expression level of I-κBα was increased significantly (P<0.05). CONCLUSION: Jiedu-Qingfei mixture may attenuate lung tissue inflammation caused by MP through NF-κB and p38 MAPK pathways.     

Role of TLR4-TRPC6 in LPS-induced NF-κB P65 expression and nuclear translocation in airway epithelial 16HBE cells

AIM: To investigate the role of Toll-like receptor 4 (TLR4) and transient receptor potential channel 6 (TRPC6) signaling pathway in lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) P65 expression and nuclear translocation in airway epithelial cells (16HBE) for supplementing the mechanism for airway inflammation. METHODS: After stimulating the 16HBE cells with LPS at 1 mg/L for 0, 0.5, 2, 6, 12 and 24 h, the expression of NF-κB P65 at mRNA and protein levels in the 16HBE cells were determined by RT-PCR and Western blot respectively, and the nuclear translocation of NF-κB P65 was detected by immunocytochemical staining method. The effects of TLR4 inhibitor CLI-095 at 5 μmol/L and TRPC6 agonist Hyp9 at 10 μmol/L on LPS (1 mg/L)-induced NF-κB P65 expression and nuclear translocation in the 16HBE cells were determined by RT-PCR, Western blot and immunocytochemical staining. RESULTS: LPS increased the mRNA and protein expression of NF-κB P65 and nuclear translocation in the 16HBE cells(P<0.05). TLR4 inhibitor CLI-095 reduced the mRNA and protein expression of NF-κB P65 and nuclear translocation induced by LPS, while Hyp9 enhanced the mRNA and protein expression of NF-κB P65 and nuclear translocation induced by LPS in the 16HBE cells(P<0.05). CONCLUSION: LPS induces the expression and nuclear translocation of NF-κB P65 in the 16HBE cells via TLR4-TRPC6 signaling pathway.     

Effect of HMGA2 gene on high glucose-induced apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway

AIM:To investigate the effect of HMGA2 down-regulation on apoptosis and Notch signaling pathway in renal tubular epithelial cells exposed to high glucose (HG). METHODS:D-glucose at 5, 10, 20 and 30 mmol/L was used to stimulate human renal tubular epithelial HK-2 cells for 2 h, and D-glucose at 30 mmol/L was used to stimulate the HK-2 cells for 10 min, 60 min and 120 min. The protein expression of HMGA2 was determined by Western blot. The HK-2 cells were divided into normal glucose (NG) group, HG group, HG+si-HMGA2 group and HG+NC group, in which siRNA was transfected by Lipofectamine^TM 2000 for 48 h. Flow cytometry was used to analyze the apoptotic rate, reactive oxygen species (ROS) assay kit was used to detect ROS content, and Western blot was used to detect the protein levels of Notch1, Hes1 and Bcl-2. The HK-2 cells were treated with the Notch signaling pathway inhibitor DAPT, and then the cells were divided into HG group, HG+DAPT group and HG+si-HMGA2+DAPT group. The apoptotic rate was analyzed by flow cytometry. RESULTS:Exposure of the HK-2 cells to D-glucose at different concentrations for different time significantly increased the expression of HMGA2 (P<0.05). Compared with NG group, the protein expression of HMGA2, Notch1 and Hes1 in HG group was increased, the expression of Bcl-2/Bax was decreased, the apoptotic rate was increased, and the content of ROS was increased obviously (P<0.05). Compared with HG group, the protein expression of HMGA2, Notch1 and Hes1 of HG+si-HMGA2 group was decreased, the expression of Bcl-2/Bax was increased, the apoptotic rate was decreased, and the content of ROS was decreased significantly (P<0.05). The apoptotic rate in HG+DAPT group was significantly lower than that in HG group, while the apoptotic rate in HG+si-HMGA2+DAPT group was significantly lower than that in HG+DAPT group (P<0.05). CONCLUSION:Down-regulation of HMGA2 expression inhibits the apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway and decreasing ROS production.     

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.     

Everolimus prevents experimental IgA nephropathy in rats

AIM: To investigate the effects of everolimus on the experimental IgA nephropathy in rats and its possible mechanisms.METHODS: The rat model of experimental IgA nephropathy was established. The rats were randomly divided into control group, IgA group and everolimus treatment group. After the corresponding treatments were given, urinary red blood cells, protein and N-acetyl-β-D-glucosaminidase (NAG) were examined. Immunofluorescence staining was used to analyze the level of IgA precipitation in the renal tissues. Additionally, the protein expression of myeloid differentiation factor 88 (MyD88), TLR4, NF-κB, IL-4 and IL-13 was determined by Western blot. The mRNA levels of IL-4 and IL-13 were detected by qPCR.RESULTS: Everolimus significantly inhibited the increases in the urinary levels of red blood cells, protein and NAG in experimental IgA nephropathy rats. Furthermore, IgA nephropathy-induced increases in the protein expression of MyD88, TLR4, NF-κB, IL-4 and IL-13 were attenuated after everolimus treatment. Similar results were obtained in the mRNA levels of IL-4 and IL-13 by qPCR detection.CONCLUSION: Everolimus improves the impairments of renal function in experimental IgA nephropathy rats as evidenced by decreasing urinary red blood cells, protein and NAG, which may be related to the inhibition of MyD88, TLR4, NF-κB, IL-4 and IL-13 expression.     

Qishen-Yiqi dripping pills attenuate atherosclerosis by regulating Ca2+ homeostasis via TRPC1/STIM1 pathway

AIM:To explore the therapeutic effect of Qishen-Yiqi dripping pills (QS) on atherosclerosis (AS) and the mechanism. METHODS:AS rat model was established by high-fat diet, and SD rats were randomly divided into normal control group, AS model group, low-dose, middle-dose and high-dose QS groups, and positive group (n=6 each). After administration for 12 weeks, serum samples were collected to detect the serum lipid and Ca²⁺ levels. HE staining was used evaluated the histopathological changes of arterial tissue. The serum levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were measured by ELISA. The nitric oxide (NO) level was detected by nitrate reductase method. The protein levels of transient receptor potential channel protein 1 (TRPC1), stromal interaction molecule 1 (STIM1) and endothelial NO synthase (eNOS) were determined by Western blot. RESULTS:QS significantly reduced the arterial damage via inhibiting the formation of atherosclerotic plaque and attenuated intimal thickening and vascular stenosis. Compared with AS group, the serum levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) were decreased significantly and the levels of high-density lipoprotein cholesterol (HDL-C) were increased significantly in high-dose QS group (P<0.05). The serum levels of IL-1β, IL-6 and TNF-α in high-dose QS group were lower than those in AS group (P<0.05). Compared with AS group, the serum Ca²⁺ level was lowered and the arterial tissue NO level was elevated in QS groups (P<0.05). Compared with AS rats, the protein levels of TRPC1 and STIM1 were decreased significantly and the protein level of eNOS was increased significantly in the rats treated with QS (P<0.05). CONCLUSION:QS regulate calcium homeostasis via TRPC1/STIM1 pathway, increase the production of NO and inhibit the inflammatory responses, thus exerting anti-AS effect.     

Effects of metformin combined with Ge Xia Zhu Yu decoction on TLR4/NF-κB signaling pathway in rats with polycystic ovary and insulin resis-tance

AIM: To study the effect of metformin (Met) combinated with Ge Xia Zhu Yu decoction on Toll-like receptor-4 (TLR-4)/nuclear factor-κB (NF-κB) signaling pathway in the rats with polycystic ovary syndrome (PCOS) and insulin resistance induced by dehydroepiandrosterone, and to explore the mechanisms. METHODS: PCOS rats (after induced by dehydroepiandrosterone, n=110) were randomly divided into 3 groups:model group (30 rats), Met treatment group (40 rats) and Met combinated with Ge Xia Zhu Yu decoction treatment (combination) group (40 rats). The rats in model group were given the same volume of 0.9% sodium chloride daily by gavage. The rats in Met group were given Met (270 mg·kg^-1·d^-1) by gavage. The rats in combination group were given Met (270 mg·kg^-1·d^-1) and Ge Xia Zhu Yu decoction (34.5 mg·kg^-1·d^-1) by gavage. All rats were continuously intervened for 28 d. After the intervention, blood glucose[fasting plasma glucose (FPG) and 2-hour postprandial blood glucose (2hPBG)] was measured. The mRNA expression levels of follicular epithelial NF-κB, TLR-4 and oxidized low-density lipoprotein (ox-LDL) were detected by RT-PCR. The serum levels of inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and C-reactive protein (CRP) were also detected by ELISA. RESULTS: After the intervention, FPG, 2hPBG, and serum levels of IL-6, TNF-α and CRP in Met group and combination group were lower than those in model group (P<0.05), and those in combination group were lower than those in Met group (P<0.05). Meanwhile, the mRNA expression levels of follicular epithelial NF-κB, TLR-4 and ox-LDL in Met group and combination group were lower than those in model group (P<0.05), and those in combination group were lower than those in Met group (P<0.05). CONCLUSION: Treatment with Met combined with Ge Xia Zhu Yu decoction improves insulin resistance in PCOS rats by decreasing the levels of inflammatory factors in serum and epithelial cells of ovary and inhibiting the expression of NF-κB, TLR-4 and ox-LDL in epithelial tissue of ovary.     

Resveratrol attenuates inflammatory pain induced by complete Freund's adjuvant via NF-κB signaling pathway in a mouse model

AIM: To investigate the anti-inflammatory action of resveratrol (Res) and its correlation with nuclear factor-κB (NF-κB) signaling pathway in a mouse model of inflammatory pain.METHODS: BALB/c mice (n=60) were randomly divided into 6 groups:normal control group, inflammatory pain model group, positive control (dexamethasone, 0.5 mg/kg) group and resveratrol (100, 50 and 25 mg/kg) groups (10 mice in each group). In order to observe the anti-inflammatory pain effects of reseratrol on mice, the paw withdrawal mechanical threshold, paw withdrawal thermal latency and cold withdrawal times were detected. In order to analyze the mechanism of analgesic effect of resveratrol, the expression levels of NF-κB, inhibitor of NF-κB (IκB) α, inhibitor of NF-κB kinase (IKK) β, tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the spinal cord tissues (L4~L6) of the mice were determined by RT-PCR and Western blot.RESULTS: The resveratrol at 100 and 50 mg/kg increased the paw withdrawal mechanical threshold, prolonged the paw withdrawal thermal latency, and decreased the cold withdrawal times in the inflammatory pain mice (P<0.05 or P<0.01). The resveratrol at 100 mg/kg down-regulated the mRNA and protein expression levels of NF-κB, IκBα, IKKβ, TNF-α and IL-1β in the spinal cord tissues (L4~L6) of inflammatory pain mice (P<0.05 or P<0.01).CONCLUSION: Resveratrol ameliorates the inflammatory pain of the mice induced by complete Freund's adjuvant. The mechanism is related to the inhibition of NF-κB signaling pathway.     

Expression and significance of TLR4/NF-κB in pulmonary vascular remodeling in smoking rats

ATM: To observe the expression of Toll-like receptor 4 (TLR4), nuclear factor-κB subunit P65 protein (NF-κB P65) and proliferating cell nuclear antigen (PCNA) in the pulmonary vascular tissues of the rats exposed to smoke, and to explore the possible mechanism of TLR4/NF-κB signaling pathway in pulmonary vascular remodeling. METHODS: SPF male healthy rats (n=48) were randomly divided into control group, smoke exposure for 4 weeks group (S4 group), smoke exposure for 8 weeks group (S8 group) and smoke exposure for 12 weeks group (S12 group), with 12 rats in each group. HE staining was used to observe the morphological changes of pulmonary vessels, and then the pulmonary vascular wall area/total vascular area (WA%) and vascular wall thickness/vascular external diameter (WT%) were measured by the medical image analysis system. The expression of TLR4, NF-κB P65 and PCNA in the pulmonary vascular tissues was detected by immunohistochemical staining. The protein content was expressed by the average integral absorbance. The mRNA expression of TLR4 in the pulmonary vessels was detected by RT-qPCR. The relationships between WA%, WT%,TLR4 protein, TLR4 mRNA, P65 protein, PCNA protein and pulmonary vascular remodeling, and another relationships between WA%, WT%, P65 protein, PCNA protein and TLR4 protein were analyzed.RESULTS: The WA% and WT% in smoke exposure groups significantly increased compared with control group, and the ratio was proportional to the time of smoke exposure. The protein expression of TLR4, p65 and PCNA, and the mRNA expression of TLR4 in smoke exposure groups also increased significantly compared with control group. CONCLUSION: The extent of pulmonary vascular remodeling in the rats increases when the protein expression of TLR4 is up-regulated. There is a positive correlation between pulmonary vascular remodeling and the protein expression of TLR4 and NF-κB P65. Pulmonary vascular remodeling may be related to the activation of TLR4/NF-κB signaling pathway.     

Inhibitory effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease rats via SIRT1/NF-κB signaling pathway

AIM: To investigate the effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease (AD) rats via SIRT1/NF-κB signaling pathway and its mechanism. METHODS: AD rat model was established by intragastric administration of AlCl₃ and intraperitoneal injection of D-galactose. After treated with butylphthalide at 25 mg/kg (low dose), 50 mg/kg (medium dose) and 100 mg/kg (high dose), the effects of butylphthalide on the morphology of hippocampal neurons, apoptosis rate, and the protein levels of Bcl-2, Bax, cleaved caspase-3 and the SIRT1/NF-κB signaling pathway associated proteins were determined by HE staining, flow cytometry and Western blot, respectively. After treated with SIRT1 agonist SRT1720 and inhibitor sirtinol, the role of SIRT1/NF-κB signaling pathway in hippocampal neuronal apoptosis was observed. On the basis of giving 50 mg/kg butylphthalide, sirtinol was administered, and the effect of butylphthalide on neuronal apoptosis regulated by SIRT1/NF-κB signaling pathway was evaluated. RESULTS: The morphology of hippocampal neurons in the AD rats were improved, the apoptosis rate of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited, and the protein levels of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted by butylphthalide significantly (P<0.05). The protein expression of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted, and the apoptosis of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited by SRT1720 remarkably (P<0.05), whereas the effect of sirtinol was contrary to that of SRT1720. After sirtinol treatment, the inhibitory effect of butylphthalide on apoptosis of hippocampal neurons, the protein levels of Bax and cleaved caspase-3, and the promotion of Bcl-2 protein expression in hippocampal neurons were markedly weakened (P<0.05). CONCLUSION: Butylphthalide inhibits the apoptosis of hippocampal neurons in the AD rats by down-regulating the protein expression of Bax and cleaved caspase-3, and up-regulating the protein expression of Bcl-2 through activating SIRT1/NF-κB signaling pathway.     

Effects of vinpocetine on inflammation of brain in rats with intracerebral hemorrhage

AIM: To investigate the effects of vinpocetine on inflammation of brain in intracerebral hemorrhage (ICH) rats and to explore the underlying mechanisms. METHODS: All rats were randomly divided into sham group, ICH group, ICH with low dose of vinpocetine treatment group, ICH with medium dose of vinpocetine treatment group, and ICH with high dose of vinpocetine treatment group. Except sham group, the rats in other groups were injected with type VⅡ collagenase to establish ICH model, and then the rats in vinpocetine treatment groups were received vinpocetine at 0.5, 1.0 or 1.5 mg/kg by intraperitoneal injection once a day for 7 days. After corresponding treatment, the impairment of neurological function in the rats was scored and the water content of the brain tissues was measured. Moreover, the activity of myeloperoxidase (MPO) was determined by ELISA, and the protein expression of Toll-like receptors 4 (TLR4), nuclear factor-κB(NF-κB), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molcule-1 (VCAM-1) in the brain tissues was determined by Western blot. RESULTS: Compared with ICH group, vinpocetine treatment significantly decreased the scores of the impairment of neurological function and the water content of the brain tissues. Moreover, the activity of MPO and the protein expression of TLR4, NF-κB, ICAM-1 and VCAM-1 were also reduced after vinpocetine treatment (P<0.05). CONCLUSION: Vinpocetine improves neurological function in ICH rats via suppression of inflammation by inhibiting NF-κB signaling and expression of ICAM-1 and VCAM-1.     

Effect of Shaofu-Zhuyu decoction on MAPK/ERK signaling pathway in rats with endometriosis

AIM: To observe the effects of Shaofu-Zhuyu decoction (SFZY) on mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway in the rats with endometriosis (EM), and to explore the mechanism of SFZY for treatment of EM.METHODS: Healthy female SD rats were used to establish the EM model. The rats were randomly divided into blank control group, model group, positive control group, and low dose, middle dose and high dose of SFZY groups. The pathological changes of the endometriotic tissue were observed by HE staining. The levels of tumor necrosis factor-α (TNF-α), interleukin-6(IL-6) and IL-8 in the uterine tissue were detected by ELISA. The mRNA expression of ERK, vascular endothelial growh factor (VEGF) and matrix metalloprotein-9 (MMP-9) was detected by RT-qPCR. The protein expression of nuclear factor-κB (NF-κB), MAPK and MAPK-ERK kinase (MEK) was determined by Western blot.RESULTS: Compared with model group, the levels of TNF-α, IL-6 and IL-8 in the uterine tissue of the rats in middle dose and high dose of SFZY groups were significantly decreased (P<0.05), the mRNA expression of ERK, VEGF and MMP-9 was significantly reduced, and the protein expression of NF-κB, MEK and MAPK was decreased significantly in the rat endometriotic tissues (P<0.05).CONCLUSION: SFZY may play a key role in the treatment of EM by regulating MAPK/ERK signaling pathway.     

Asiaticoside attenuates hypoxic pulmonary hypertension in mice by inhibiting NF-κB/p38 signaling pathway

AIM: To investigate whether asiaticoside attenuates hypoxic pulmonary hypertension by inhibiting p38/NF-κB signaling pathway. METHODS: BALB/c mice (n=30) were randomly divided into normoxia (N) group, hypoxia (H) group, and hypoxia+asiaticoside group. Right ventricular systolic pressure (RVSP), mean carotid artery pressure (mCAP), the weight ratio of right ventricle/(left ventricle+ventricular septum)[RV/(LV+S)], the ratio of right ventricle/body weight (RV/BW), vessel wall area/vessel total area (WA/TA) and vessel wall diameter/vessel wall total diameter (WT/TT) were determined after the model was established. The protein levels of p38, p-p38, NF-κB and p-NF-κB in the lung tissues were detected by Western blot. The fluorescence intensity of p-p38 and p-NF-κB were measured by immunofluorescence method. The serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA. RESULTS: Compared with N group, the levels of RVSP, RV/(LV+S), RV/BW, WA/TA and WT/TT were significantly increased in H group, while administration of asiaticoside decreased the levels of RVSP, RV/(LV+S), RV/BW, WA/TA and WT/TT (P<0.05). Compared with N group, the relative protein levels of p-p38 and p-NF-κB in H group were significantly increased (P<0.05), and the concentrations of IL-6 and TNF-α were significantly increased, which were apparently attenuated by asiaticoside injection. CONCLUSION: Inhibition of p38/NF-κB signaling pathway and reduction of inflammatory responses may be the important mechanisms of asiaticoside in the prevention and treatment of hypoxic pulmonary hypertension.     

Expression of calprotectin in rats with renal ischemia-reperfusion injury

AIM: To investigate the expression of calprotectin(CALP) in the rats with renal ischemia-reperfusion injury(IRI). METHODS: Male Sprague-Dawley rats were randomly divided into sham operation and IRI group(n=25 in each group). Blood samples and the kidneys were obtained at 6 h, 12 h, 24 h, 48 h and 72 h after reperfusion. The pathological changes of the kidneys were observed. The serum concentrations of blood urea nitrogen(BUN) and serum creatinine(SCr) were measured. The serum levels of CALP, tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) were detected by ELISA, and the expression of CALP, Toll-like receptor 4(TLR4) and NF-κB p65 in the renal tissues were determined by the methods of immunohistochemistry and Western blot. RESULTS: Different serial ischemia changes were observed in the renal tissues, mainly in the renal tubular epithelial cells and the mesenchyma, with the infiltration of inflammatory cells. The serum levels of BUN, SCr, CALP, TNF-α and IL-6 in IRI group were markedly increased as compared with sham group(P<0.05). The protein expression of CALP, TLR4 and NF-κB p65 in the renal tubular epithelial cells in IRI group was greatly enhanced in comparison with that in sham group(P<0.05). CONCLUSION: The serum concentrations of CALP, TNF-α and IL-6, and the protein expression levels of CALP, TLR4 and NF-κB p65 in the renal tissue are significantly increased in the rats with IRI, suggesting that calprotectin plays an important role in the inflammation in rats with IRI.     

Effect of salidroside on alcohol-injuced hepatic injury in rats

AIM:To investigate the effect of salidroside on alcoholic hepatic injury in rats. METHODS:The SD rats were randomly divided into 5 groups:negative control group, model group, bifendate group, and low-and high-dose salidroside groups. The rats in model group were administered with 56% alcohol, while the rats in negative control group was administered with saline. The rats in bifendate group and salidroside groups were administered with corresponding drugs every day. The blood and the liver tissues were collected to measure triacylglycerol (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA) and superoxide dismutase (SOD). The pathological changes of the liver tissues were observed with HE staining. Tumor necrosis factor-α (TNF-α) and nuclear factor-κB (NF-κB) were measured by ELISA and the protein and mRNA expression levels of TNF-α and NF-κB were detected by Western blot and RT-PCR. RESULTS:Compared with model group, the levels of TG, ALT, AST, MDA, TNF-α and NF-κB were reduced, while the activity of SOD was enhanced in salidroside group (P<0.05). The liver tissue injury was significantly attenuated. CONCLUSION:Salidroside improves the pathological changes, reduces inflammation, increases the activity of antioxidant enzymes and reduces lipid peroxidation in the liver with alcohol-induced injury. This effect may be related to regulating the NF-κB-mediated inflammatory responses.