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OBJECTIVE@#To evaluate the effect of curcumin on renal mitochondrial oxidative stress, nuclear factor-κB/NOD-like receptor protein 3 (NF-κB/NLRP3) inflammatory body signaling pathway and tissue cell injury in rats with acute respiratory distress syndrome (ARDS).@*METHODS@#A total of 24 specific pathogen free (SPF)-grade healthy male Sprague-Dawley (SD) rats were randomly divided into control group, ARDS model group, and low-dose and high-dose curcumin groups, with 6 rats in each group. The ARDS rat model was reproduced by intratracheal administration of lipopolysaccharide (LPS) at 4 mg/kg via aerosol inhalation. The control group was given 2 mL/kg of normal saline. The low-dose and high-dose curcumin groups were administered 100 mg/kg or 200 mg/kg curcumin by gavage 24 hours after model reproduction, once a day. The control group and ARDS model group were given an equivalent amount of normal saline. After 7 days, blood samples were collected from the inferior vena cava, and the levels of neutrophil gelatinase-associated lipocalin (NGAL) in serum were determined by enzyme-linked immunosorbent assay (ELISA). The rats were sacrificed, and kidney tissues were collected. Reactive oxygen species (ROS) levels were determined by ELISA, superoxide dismutase (SOD) activity was detected using the xanthine oxidase method, and malondialdehyde (MDA) levels were determined by colorimetric method. The protein expressions of hypoxia-inducible factor-1α (HIF-1α), caspase-3, NF-κB p65, and Toll-like receptor 4 (TLR4) were detected by Western blotting. The mRNA expressions of HIF-1α, NLRP3, and interleukin-1β (IL-1β) were detected by reverse transcription-polymerase chain reaction (RT-PCR). Renal cell apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL). The morphological changes in renal tubular epithelial cells and mitochondria were observed under a transmission electron microscope.@*RESULTS@#Compared with the control group, the ARDS model group exhibited kidney oxidative stress and inflammatory response, significantly elevated serum levels of kidney injury biomarker NGAL, activated NF-κB/NLRP3 inflammasome signaling pathway, increased kidney tissue cell apoptosis rate, and renal tubular epithelial cell damage and mitochondrial integrity destruction under transmission electron microscopy, indicating successful induction of kidney injury. Following curcumin intervention, the injury to renal tubular epithelial cells and mitochondria in the rats was significantly mitigated, along with a noticeable reduction in oxidative stress, inhibition of the NF-κB/NLRP3 inflammasome signaling pathway, and a significant decrease in kidney tissue cell apoptosis rate, demonstrating a certain dose-dependency. Compared with the ARDS model group, the high-dose curcumin group exhibited significantly reduced serum NGAL levels and kidney tissue MDA and ROS levels [NGAL (μg/L): 13.8±1.7 vs. 29.6±2.7, MDA (nmol/g): 115±18 vs. 300±47, ROS (kU/L): 75±19 vs. 260±15, all P < 0.05], significantly down-regulated protein expressions of HIF-1α, caspase-3, NF-κB p65, and TLR4 in the kidney tissue [HIF-1α protein (HIF-1α/β-actin): 0.515±0.064 vs. 0.888±0.055, caspase-3 protein (caspase-3/β-actin): 0.549±0.105 vs. 0.958±0.054, NF-κB p65 protein (NF-κB p65/β-actin): 0.428±0.166 vs. 0.900±0.059, TLR4 protein (TLR4/β-actin): 0.683±0.048 vs. 1.093±0.097, all P < 0.05], and significantly down-regulated mRNA expressions of HIF-1α, NLRP3, and IL-1β [HIF-1α mRNA (2-ΔΔCt): 2.90±0.39 vs. 9.49±1.87, NLRP3 mRNA (2-ΔΔCt): 2.07±0.21 vs. 6.13±1.32, IL-1β mRNA (2-ΔΔCt): 1.43±0.24 vs. 3.95±0.51, all P < 0.05], and significantly decreased kidney tissue cell apoptosis rate [(4.36±0.92)% vs. (27.75±8.31)%, P < 0.05], and significantly increased SOD activity (kU/g: 648±34 vs. 430±47, P < 0.05).@*CONCLUSIONS@#Curcumin can alleviate kidney injury in ARDS rats, and its mechanism may be related to the increasing in SOD activity, reduction of oxidative stress, and inhibition of the activation of the NF-κB/NLRP3 inflammasome signaling pathway.
Subject(s)
Male , Rats , Animals , Rats, Sprague-Dawley , NF-kappa B , Actins , Caspase 3 , Curcumin , Lipocalin-2 , Toll-Like Receptor 4 , Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Reactive Oxygen Species , Saline Solution , Kidney , Superoxide DismutaseABSTRACT
Objective:To study the analgesic effect of α-cobratoxin (α-CbTX) on mice and its effect on protein kinase A (PKA) activity of spinal dorsal root ganglion (DRG) in mice.Methods:Healthy male ICR mice( n=102) were randomly divided into low-, medium-, and high-dose α-CbTX groups (1 mg/kg, 3 mg/kg, 9 mg/kg respectively, gavage, n=21), solvent control group (equivalent volume of 0.9% normal saline, gavage, n=21), morphine positive control group (3 mg/kg, intraperitoneal injection, n=6)or aspirin positive control group(300 mg/kg, gavage, n=12). The analgesic effect of α-CbTX was evaluated by hot plate test, acetic acid twisting test and formalin foot licking test. Formalin plantar injection was used to induce pain and then the L4-L6 DRG was taken 30 minutes later. The expression of PKA C-α in L4-L6 DRG of mice were detected by Western blot.SPSS 16.0 software was used for statistical analysis. Repeated measurement ANOVA was used to evaluate the hot plate experimental data, and one-way ANOVA was used for other experimental data. LSD- t test was used for further pairwise comparison. Results:In the hot plate test, the interaction between group and time of mice paw licking latency was significant ( F=8.902, P<0.05). At 0.5 h after administration, the paw licking latencies of α-CbTX medium-dose group ((11.83±1.47)s)and α-CbTX high-dose group (( 14.33±12.1)s) were both longer than that of solvent control group((8.17±0.75) s) ( t=4.461, 7.053, both P<0.05). The efficacy of α-CbTX medium dose group lasted until 1.5 h after administration (all P<0.05), and that of α-CbTX high dose group lasted until 2 h after administration(all P<0.05). In the acetic acid writhing test, the writhing times in the low-, medium- and high-dose α-CbTX group((34.50±3.62) times, (26.17±2.40) times, (13.83±3.76) times)) were significantly lower than that in solvent control group ((42.50±4.59) times) ( t=3.938, 8.040, 14.112, all P<0.05). In the period of the formalin test phase Ⅱ, the total licking time of α-CbTX low-, medium- and high-dose groups ((71.17±6.46) s), (54.67±6.41) s, (40.50±3.89)s) were significantly shorter than that of the solvent control group ((98.67±11.50) s)( t=6.950, 11.120, 14.700, all P<0.05). In the Western blot experiment, compared with solvent control group (0.22±0.01), the levels of PKA C-α in the DRG of mice in low-, medium- and high-dose α-CbTX groups ((0.31±0.02), (0.41±0.03), (0.44±0.02)) were up-regulated ( t=3.140, 6.471, 7.492, all P<0.05). Conclusion:α-CbTX has obvious analgesic effect, and its analgesic mechanism may be related to the activation of PKA.
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Objective:To investigate the effect of curcumin on renal fibrosis in lipopolysaccharide (LPS) induced acute respiratory distress syndrome (ARDS) in adult SD rats.Methods:Twenty-four male SD rats were randomly (random number) divided into four groups: control group, ARDS group, low dose group, and high dose group ( n=6 per group). In the control group, the rats were given atomization intratracheal of standard saline 2 mL/kg; in the ARDS group, low-dose group, and high-dose group, the rats were given atomization intratracheal of 4 mg/kg LPS; in the low-dose group, the rats were given curcumin 100 mg/d by the oral administration, and in the high-dose group, the rats were given curcumin or 200 mg/d respectively. After seven days, the rats were sacrificed. The superoxide dismutase (SOD) activity and the content of malondialdehyde (MDA) and glutathione (GSH) in renal tissue were detected by colorimetric assay. Nuclear factor kappa-B p65 (NF-κB p65) and transforming growth factor-β1 (TGF-β1) were detected by Western blot. The expression of interleukin-6 (IL-6) mRNA, proline hydroxylase 3 (PHD3) mRNA, vascular endothelial growth factor (VEGF) mRNA and erythropoietin receptor (EPOR) mRNA were detected by quantitative real-time PCR (qRT-PCR). HE staining and Masson staining were used to assess pathological damage. One-way analysis of variance was used for comparison among multiple groups and SNK method was used for comparison between two groups. Results:Compared with the control group, the SOD activity and GSH content in the ARDS group, low-dose group, and high-dose group were significantly decreased (all P<0.05); the protein expression levels of MDA, NF-κB p65, and TGF-β1 were increased significantly, and IL-6 mRNA, PHD3 mRNA, VEGF mRNA, and EPOR mRNA were significantly upregulated (all P<0.05). HE staining showed inflammatory cell infiltration, and fibrogenesis in kidney tissue, and Masson staining showed deposition of collangen-like substance. Compared with the ARDS group, SOD activity and GSH content were increased, while the protein expression of NF-κB p65 and TGF-β1, IL-6 mRNA, PHD3 mRNA, VEGF mRNA, and EPOR mRNA were decreased significantly in the low-dose group and high-dose group (all P<0.05). Curcumin therapy reduced inflammatory cellular infiltration, and the deposition of collagen-like substance in kidney tissue. Compared with the low-dose group, SOD activity and GSH content were increased in the high-dose group (all P<0.05), and the protein expression of NF-κB p65 and TGF-β1, IL-6 mRNA, PHD3 mRNA, VEGF mRNA, and EPOR mRNA were decreased significantly in the high-dose group (all P<0.05). The high-dose group exhibited a significant reduction in edema, and a decrease of the deposition of collagen-like substance in kidney tissue. Conclusions:Curcumin can inhibit the development of renal fibrosis induced by acute respiratory distress syndrome in rats, and its mechanism may be related to inhibiting the expression of inflammatory factors and enhancing hypoxia tolerance.
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Objective To study the therapeutic effect of anti-CD80 bivalent antibody on mouse lu-pus nephritis and to explore the possible molecular mechanism. Methods A mouse model of lupus nephritis was established through intraperitoneal injection of 0. 5 ml of pristine in female C57BL/6J mice. Appearance of urinary protein and significantly increased levels of peripheral antinuclear antibody ( ANA) and anti-doub-le-stranded DNA ( anti-dsDNA) antibody in the fourth month after injection indicated that the mouse model was established successfully. Then the mice were divided into two groups including anti-CD80 bivalent anti-body intervention group (injected with 200μg of anti-CD80 bivalent antibody at day 1, 3, 5, 8 and 15, fol-lowed by three times of injection with an interval of one month) and model group ( injected with the same protein using the same strategy). A normal control group was set up accordingly. Albustix test paper was used to monitor the dynamic changes in mouse urinary protein. Flow cytometry was used to analyze the acti-vation of immune-related cells in spleen. Levels of autoantibodies ( ANA and anti-dsDNA) and levels of IFN-γ and IL-4 in serum were detected by indirect immunofluorescence assay. Renal tissue samples were an-alyzed with hematoxylin and eosin ( HE) staining and immunocomplex ( IC) assay. Results Urinary pro-tein level of the anti-CD80 bivalent antibody intervention group was significantly lower than that of the model group (P<0. 05). Activated macrophages, dendritic cells, neutrophils and B cells in spleen tissues of the anti-CD80 bivalent antibody intervention group were significantly less than those of the model group ( P<0. 05), and the numbers of CD4+ and CD154+ T cells were significantly less than those of the model group (P<0. 05). Positive rates and titers of ANA and dsDNA in serum samples of the intervention group were lower than those of the model group (P<0. 05). Levels of IFN-γand IL-4 in serum samples of the interven-tion group were decreased as compared with those of the model group (P<0. 05). HE staining and immuno-fluorescence assay showed that glomerular inflammatory injury and necrosis were alleviated and kidney im-mune complex deposition was reduced after anti-CD80 bivalent antibody intervention. Conclusion Anti-CD80 bivalent antibody specifically binds to the CD80 molecule on antigen presenting cell surface, blocks the CD80/CD28 co-stimulatory signaling pathway and down-regulates the body′s immune response, which al-leviates and reverses the lupus-like nephritis-induced pathological damages in mice.
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Objective To study the therapeutic effect of anti-CD80 bivalent antibody on mouse lu-pus nephritis and to explore the possible molecular mechanism. Methods A mouse model of lupus nephritis was established through intraperitoneal injection of 0. 5 ml of pristine in female C57BL/6J mice. Appearance of urinary protein and significantly increased levels of peripheral antinuclear antibody ( ANA) and anti-doub-le-stranded DNA ( anti-dsDNA) antibody in the fourth month after injection indicated that the mouse model was established successfully. Then the mice were divided into two groups including anti-CD80 bivalent anti-body intervention group (injected with 200μg of anti-CD80 bivalent antibody at day 1, 3, 5, 8 and 15, fol-lowed by three times of injection with an interval of one month) and model group ( injected with the same protein using the same strategy). A normal control group was set up accordingly. Albustix test paper was used to monitor the dynamic changes in mouse urinary protein. Flow cytometry was used to analyze the acti-vation of immune-related cells in spleen. Levels of autoantibodies ( ANA and anti-dsDNA) and levels of IFN-γ and IL-4 in serum were detected by indirect immunofluorescence assay. Renal tissue samples were an-alyzed with hematoxylin and eosin ( HE) staining and immunocomplex ( IC) assay. Results Urinary pro-tein level of the anti-CD80 bivalent antibody intervention group was significantly lower than that of the model group (P<0. 05). Activated macrophages, dendritic cells, neutrophils and B cells in spleen tissues of the anti-CD80 bivalent antibody intervention group were significantly less than those of the model group ( P<0. 05), and the numbers of CD4+ and CD154+ T cells were significantly less than those of the model group (P<0. 05). Positive rates and titers of ANA and dsDNA in serum samples of the intervention group were lower than those of the model group (P<0. 05). Levels of IFN-γand IL-4 in serum samples of the interven-tion group were decreased as compared with those of the model group (P<0. 05). HE staining and immuno-fluorescence assay showed that glomerular inflammatory injury and necrosis were alleviated and kidney im-mune complex deposition was reduced after anti-CD80 bivalent antibody intervention. Conclusion Anti-CD80 bivalent antibody specifically binds to the CD80 molecule on antigen presenting cell surface, blocks the CD80/CD28 co-stimulatory signaling pathway and down-regulates the body′s immune response, which al-leviates and reverses the lupus-like nephritis-induced pathological damages in mice.
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Objective To investigate the role of 2B subunits-containing N-methyl-D-aspartate receptors (NR2B) in the arcuate nucleus in the development of inflammatory pain (IP) in rats.Methods One hundred and eight male Sprague-Dawley rats, aged 9 weeks, weighing 200-250 g, were randomly divided into 4 groups using a random number table: sham operation group (group S, n =47);group IP (n =47);dimethyl sulfoxide control group (group DMSO, n =7);selective NR2B antagonist Ro25-6981 group (group Ro25-6981, n=7).IP was induced by injecting complete Freund's adjuvant (CFA) 0.1 ml into the plantar surface of the left hindpaw.Ro25-6981 400 pmol was injected into the arcuate nucleus at 3 days after CFA injection.Seven rats in each group were selected for measurement of the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) at 1 day before CFA injection (T1) and at 2 days after CFA injection (T2), at 30 min before administration on 3rd day (T3) , at 30 min after administration on 3rd day (T4) , and on 5th day (T5).In S and IP groups, The rats were sacrificed at T1-3 and T5 , and the arcuate nucleus of the hypothalamus was removed for determination of NR2B mRNA expression (by real-time reverse transcriptase polymerase chain reaction) and NR2B and phosphorylated NR2B (p-NR2B) expression (by Western blot).Conclusion Compared with group S, the MWT was significantly decreased, and the TWL was shortened at T2-5 in IP, DMSO and Ro25-6981 groups, and the expression of p-NR2B was up-regulated at each time point (P＜0.05) , and no significant change was found in NR2B protein and mRNA expression in group IP (P＞0.05).Compared with group IP, the MWT was significantly increased, and the TWL was prolonged at T4in group Ro25-6981 (P＜0.05) , and no significant change was found in MWT and TWL at each time point in group DMSO (P＞0.05).Conclusion The activation of NR2B in the arcuate nucleus is involved in the development of IP in rats.