The effect of glucocorticoid receptor on level of inflammatory media in rats after critical scald

AIM:To further elaborate the effect of glucocorticoid receptor(GR) decrease on level of inflammatory media in rats after critical scald.METHOD:The changes of phospholipase A₂(PLA₂) activity and concentrations of tumor necrosis factoralpha (TNFα) and malondialdehyde (MDA), the product of lipid peroxidation metabolism in plasma and tissue homogenate have been studied in scalded rats with or without GR blockade by RU38486.RESULTS:The PLA₂ activity and the concentrations of TNFα and MDA in plasma and homogenate of pulmonary and renal tissue in scalded rats were significantly higher than those in the controls(P＜0.01) and further increased in scalded rats accompanied with GR blockade (P＜0.01 or P＜0.05).CONCLUSION: Blockade of GR might aggravate the increase of inflammatory mediator caused by scalding. It suggested that GR might potentiate anti-inflammation in scald.     

CYP450 epoxygenase/EET pathway attenuates adipose inflammation of obese mice through HIF-1α

AIM To investigate the effects of cytochrome P450 （CYP450） epoxygenase/epoxyeicosatrienoic acid （EET） pathway on insulin resistance in obese mice, and to explore the possible mechanisms. METHODS High-fat diet-induced obesity model was established in C57BL/6Cnc mice, and the obese mice were randomly divided into 3 groups, including obesity group （treated with saline; n=10）, EET group （treated with 11,12-EET; n=10） and EET inhibitor 14,15-epoxyeicosa-5（Z）-enoic acid （EEZE） group （n=10）. Normal C57BL/6Cnc mice （n=10） treated with saline served as control. Protein expression of CYP2J2 （one of CYP450 epoxygenases） and hypoxia-inducible factor-1α （HIF-1α） was measured by Western blot. Vessel-like structure was detected by immunofluorescence staining. The serum levels of insulin, tumor necrosis factor-α （TNF-α）, interleukin （IL）-1β, IL-6 and monocyte chemoattractant protein-1 （MCP-1） were measured by ELISA. RESULTS In obese mice, homeostasis model assessment of insulin resistance （HOMA-IR） values were increased, the protein level of CYP2J2 was reduced, and the protein level of HIF-1α was increased in adipose tissues as compared with the controls （P<0.05）. The serum levels of MCP-1, IL-1β, IL-6 and TNF-α were also significantly increased in obese mice （P<0.05）. After treatment with 11, 12-EET, the HOMA-IR values were decreased compared with vehicle-treated obese mice, HIF-1α expression levels were decreased in the adipose tissue, and the serum levels of MCP-1, IL-1β, IL-6 and TNF-α were reduced （P<0.05）. Immunohistochemical results of adipose tissue from vehicle-treated obese mice showed a marked decrease in vessel-like structures （CD31-positive） compared with normal control mice （P<0.05）. EET treatment significantly increased the newly formed vessel-like structures in the visceral adipose tissues of obese mice as compared with vehicle-treated obese mice （P<0.05）. CONCLUSION High-fat diet-induced obesity and insulin resistance are closely related to the CYP450 pathway. Exogenous EETs effectively decrease obesity-induced insulin resistance possibly through pro-angiogenesis and attenuation of hypoxia and inflammation.     

Adrenomedullin (AM) and adrenomedullin binding protein (AM-BP) in the bovine mammary gland and milk: Effects of stage of lactation and experimental intramammary E. coli infection

Adrenomedullin (AM) has been characterized as an endogenous tissue survival factor and modulator of many inflammatory processes. Because of the increased susceptibility of the mammary gland to infection during the time surrounding parturition in the cow, we investigated how milk and tissue content of AM and its binding protein (AM-BP) might be affected by the stage of lactation and the udder health status. Milk and mammary biopsy samples were obtained from Holstein cows 21 days prior to and at various times after calving to represent the dry period and early and mid-stages of lactation. Additional cows received an intramammary challenge with Escherichia coli for immunohistochemical characterization of AM and AM-BP. Milk AM concentrations were relatively constant across the stages of lactation while AM-BP increased two-fold (P<0.04) between early and mid-lactation. Milk AM (P<0.04) and AM-BP (P<0.03) increased as somatic cell counts (SCCs) increased within a given stage of lactation. Tissue content of both (AM and AM-BP) were significantly affected by stage of lactation, lowest in the dry period and progressively increasing to peak at mid-lactation as well as increasing in association with higher levels of SCCs. Following E. coli challenge, AM increased in epithelial cells surrounding mammary alveoli presenting high levels of SCCs. The data suggest that AM and AM-BP are cooperatively regulated in the mammary gland during lactation; changes in localized tissue AM and AM-BP content reflect a dynamic regulation of these tissue factors in the bovine mammary gland consistent with their protective effects within inflamed tissue.     

Mechanism of Fuzilizhong decoction alleviating inflammatory damage of rats with non-alcoholic fatty liver disease

AIM To observe the effect of Fuzilizhong decoction on the inflammatory damage of non-alcoholic fatty liver disease （NAFLD） rats and to explore its mechanism. METHODS SPF male SD rats were randomly divided into 6 groups： control group, model group, high dose （20 mg·kg^-1·d^-1）, middle dose （10 mg·kg^-1·d^-1）, low dose （5 mg·kg^-1·d^-1） Fuzilizhong decoction group and Yishanfu （30 mg·kg^-1·d^-1）group, 8 rats in each group. A NAFLD rat modelwas established by intragastric administration of fat emulsion for 4 weeks. Then the drug was given for 4 weeks in each treatment group. HE staining was performed to observe the histopathological changes of the rat liver.The serum levels of interleukin-2（IL-2）, IL-6 and tumor necrosis factor-α（TNF-α） were measured by ELISA. The expression of toll like receptor 4（TLR4） and NF-κB p65 in liver tissues at mRNA and protein levels was determined by RT-qPCR and Western bolt,respectively. RESULTS Compared with control group, the inflammatory damage of liver tissue was more serious, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression TLR4 and NF-κB p65 in liver tissues were significantly increased in model group（P<0.05）. However, compared with model group, the liver pathological changes in each treatment group were significantly relieved, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression of TLR4 and NF-κB p65 in liver tissues were significantly reduced（P<0.05）.In addition, the changes of TLR4 and p-NF-κB p65 protein levels in liver tissue were consistent with the changes of TLR4 and NF-κB p65 mRNA. CONCLUSION Fuzilizhong decoction attenuates the inflammatory damages of NAFLD in rats by inhibiting TLR4/NF-κB p65 signaling pathway.     

Effects of autophagy inhibitor bafilomycin A1 on macrophages polarization

AIM:To investigate the effect of bafilomycin A1 (Baf A1) on polarization in mouse macrophages RAW264.7. METHODS:The macrophages RAW264.7 were treated with Baf A1, the concentration of M1/M2 polarization related cytokines were determined by ELISA. The markers of M1/M2 polarization in the macrophages were determined by flow cytometry. The formation of autophagicbody was observed by immunofluorescence. Western blot was used to detect the expression of autophagy related protein levels. RESULTS:The concentration of M1 related proinflammatory cytokine tumor necrosis factor-α (TNF-α) was increased significantly after Baf A1 intervention (P<0.01). However, the concentration of M2 related anti-inflammatory cytokines IL-10 and IL-13 showed no significant difference. The double positive rate of CD197 and HLA-DR (M1 marker) positive cells in Baf A1 treated group were significant higher than that in control group (P<0.05), indicated that Baf A1 induced polarization of macrophage to M1. The results of immunofluoresence showed that the autophagosomes formation was notable increased in Baf A1 group (P<0.05), meanwhile Western blot results showed the expression of autophay related protein LC3-Ⅱ but not P62 was marked up-regulated (P<0.05). For autophagy activator rapamycin (Rapa) treated group, autophagosome formation was also significant increased (P<0.05), but the expression of P62 was notable down-regulated (P<0.05). CONCLUSION:Baf A1 induces polarization of mouse macrophage RAW264.7 to M1, which may be related to the inhibitory effect on the formation of autolysosome.     

Involvement of TLR4/NLRP3 inflammasome in contrast medium-induced inflammation and injury in renal tubular epithelial cells

AIM: To investigate whether Toll-like receptor 4 (TLR4) and Nod-like receptor protein 3 (NLRP3) inflammasome were involved in contrast medium (CM)-induced inflammation and injury in renal tubular epithelial cells. METHODS: Iopromide was used to injure NRK-52E cells in the study. The cell viability was measured by CCK-8 assay. The protein levels of TLR4, NLRP3, apoptosis-associated speckle-like protein (ASC), caspase-1 and cleaved caspase-3 were determined by Western blot. The releases of interleukin (IL)-1β and IL-18 were detected by ELISA. The apoptotic rate was evaluated by Hoechst staining, and mitochondrial membrane potential (MMP) was analyzed by JC-1 staining. siRNA was transfected into the NRK-52E cells to silence NLRP3 expression. RESULTS: CM decreased the viability of NRK-52E cells (P<0.05). CM also elevated the protein levels of cleaved caspase-3, TLR4, NLRP3, IL-1β and IL-18 (P<0.05). Silencing NLRP3 attenuated CM-induced releases of inflammatory cytokines. Moreover, treatment with TLR4 inhibitor TAK-242 or knockdown of NLRP3 by siRNA transfection both attenuated cell apoptosis and loss of MMP caused by CM. CONCLUSION: TLR4/NLRP3 inflammasome takes part in the pathogenesis of CM-induced acute kidney injury, and mediates CM-induced injury and inflammation in renal tubular epithelial cells.     

Haptoglobin Quantitation in Serum Samples from Clinically Normal and Clinically Abnormal Horses

Haptoglobin (HP) is a common minor to moderate acute-phase protein in mammals. It has been described as increasing under a variety of conditions in horses, with the use of different assay methods. The goal of the current study was to provide updated information regarding this biomarker by using a commonly available automated assay. In the present study, reference intervals for HP were generated using 43 serum samples from clinically normal horses. The reference interval was determined via the robust method as 0.29-2.26 mg/ml. A statistically significant 3.3-fold mean increase was observed in HP levels from a clinically abnormal group of horses (n = 33). A weak but significant correlation was found between total white blood cell count and HP levels (r = 0.37, P < .05). A significantly higher level of HP expression was observed in samples acquired from patients whose clinical signs lasted for 7 days or longer than from those whose signs were 4 days or less.     

Dietary dihydroartemisinin supplementation alleviates intestinal inflammatory injury through TLR4/NOD/NF-κB signaling pathway in weaned piglets with intrauterine growth retardation

The aim of present study was to evaluate whether diets supplemented with dihydroartemisinin (DHA) could alleviate intestinal inflammatory injury in weaned piglets with intrauterine growth retardation (IUGR). Twelve normal birth weight (NBW) piglets and 12 piglets with IUGR were fed a basal diet (NBW-CON and IUCR-CON groups), and another 12 piglets with IUGR were fed the basal diet supplemented with DHA at 80 mg/kg (IUGR-DHA group) from 21 to 49 d of age. At 49 d of age, 8 piglets with similar body weight in each group were sacrificed. The jejunal and ileal samples were collected for further analysis. The results showed that IUGR impaired intestinal morphology, increased intestinal inflammatory response, raised enterocyte apoptosis and reduced enterocyte proliferation and activated transmembrane toll-like receptor 4 (TLR4)/nucleotide-binding and oligomerization domain (NOD)/nuclear factor-κB (NF-κB) signaling pathway. Dihydroartemisinin inclusion ameliorated intestinal morphology, indicated by increased villus height, villus height-to-crypt depth ratio, villus surface area and decreased villus width of piglets with IUGR (P < 0.05). Compared with NBW piglets, IUGR piglets supplemented with DHA exhibited higher apoptosis index and caspase-3 expression, and lower proliferation index and proliferating cell nuclear antigen expression in the intestine (P < 0.05). Dihydroartemisinin supplementation attenuated the intestinal inflammation of piglets with IUGR, indicated by increased concentrations of intestinal inflammatory cytokines and lipopolysaccharides (P < 0.05). In addition, DHA supplementation down-regulated the related mRNA expressions of TLR4/NOD/NF-κB signaling pathway and upregulated mRNA expressions of negative regulators of TLR4 and NOD signaling pathway in the intestine of piglets with IUGR (P < 0.05). Piglets in the IUGR-DHA group showed lower protein expressions of TLR4, phosphorylated NF-κB (pNF-κB) inhibitor α, nuclear pNF-κB, and higher protein expression of cytoplasmic pNF-κB in the intestine than those in the IUGR-CON group (P < 0.05). In conclusion, DHA supplementation could improve intestinal morphology, regulate enterocyte proliferation and apoptosis, and alleviate intestinal inflammation through TLR4/NOD/NF-κB signaling pathway in weaned piglets with IUGR.