Case Of Injection Research Articles

Decreasing effects of protein kinase inhibitors on the expression of NOS2 and inflammatory cytokines and on phagocytosis in rat peritoneal macrophages is partly related to repolarization

The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) pathway plays a pivotal role in macrophage polarization, but other signaling routes may also be involved. The aim of this study was to reveal the relationship of activation between rat peritoneal macrophages and their polarization, to detect the signaling routes involved, and find selective protein kinase inhibitors decreasing the production of inflammatory proteins in activated peritoneal macrophages. Rat macrophages were elicited with i.p. casein injection. CD80 and CD206 markers, NOS2 (Nitric oxide synthase 2), arginase, cytokines and phagocytosis were investigated by ELISA (Enzyme Linked Immunosorbent Assay), Western Blot, fluorescent microscopic and flow cytometry. Statistical methods were ANOVA (Analysis Of Variance) and Student t-tests. Resident and elicited cells expressed both CD80 and CD206 polarization markers. The involvement of MAPK (mitogen-activated protein kinases) and JAK/STAT pathways in the polarization was evidenced by a phosphorylation array, supported by Western blotting, by cytokine markers and by the inhibitory effects of kinase inhibitors. The expression of NOS2 and inflammatory cytokines was higher in elicited cells suggesting their M1 polarization. This effect was reduced by the inhibitors of MAPK and JAK/STAT pathways. Phagocytosis was also higher in elicited macrophages and decreased by these inhibitors. Nevertheless, they cannot change macrophage polarization unambiguously, as levels of CD80 and CD206 markers were not changed. For comparison, human blood macrophages were also studied. Similar effects and several differences were observed between the two types of macrophages, suggesting the role of the previous differentiation in defining their characteristics. Selected anti-cancer protein kinase inhibitors of p38, MAPK and JAK/STAT pathways are possible candidates for the therapy of inflammatory diseases.

Fever During Localized Inflammation in Mice Is Elicited by a Humoral Pathway and Depends on Brain Endothelial Interleukin-1 and Interleukin-6 Signaling and Central EP3 Receptors.

We examined the signaling route for fever during localized inflammation in male and female mice, elicited by casein injection into a preformed air pouch. The localized inflammation gave rise to high concentrations of prostaglandins of the E species (PGE2) and cytokines in the air pouch and elevated levels of these inflammatory mediators in plasma. There were also elevated levels of PGE2 in the cerebrospinal fluid, although there was little evidence for PGE2 synthesis in the brain. Global deletion of the PGE2 prostaglandin Ereceptor3 (EP3) abolished the febrile response as did deletion of the EP3 receptor in neural cells, whereas its deletion on peripheral nerves had no effect, implying that PGE2 action on this receptor in the CNS elicited the fever. Global deletion of the interleukin-1 receptor type 1 (IL-1R1) also abolished the febrile response, whereas its deletion on neural cells or peripheral nerves had no effect. However, deletion of the IL-1R1 on brain endothelial cells, as well as deletion of the interleukin-6 receptor α on these cells, attenuated the febrile response. In contrast, deletion of the PGE2 synthesizing enzymes cyclooxygenase-2 and microsomal prostaglandin synthase-1 in brain endothelial cells, known to attenuate fever evoked by systemic inflammation, had no effect. We conclude that fever during localized inflammation is not mediated by neural signaling from the inflamed site, as previously suggested, but is dependent on humoral signaling that involves interleukin actions on brain endothelial cells, probably facilitating PGE2 entry into the brain from the circulation and hence representing a mechanism distinct from that at work during systemic inflammation.

A high-fat, high-protein diet attenuates the negative impact of casein-induced chronic inflammation on testicular steroidogenesis and sperm parameters in adult mice

The interaction between obesity and chronic inflammation has been studied. Diet-induced obesity or chronic inflammation could reduce the testicular functions of males. However, the mechanism underlying the reproductive effects of fattening foods in males with or without chronic inflammation still needs further discussion. This study was aimed to investigate the effects of high-fat, high-protein diet on testicular steroidogenesis and sperm parameters in adult mice under physiological and chronic inflammatory conditions. Because casein can trigger a non-infectious systemic inflammatory response, we used casein injection to induce chronic inflammation in male adult Kunming mice. Twenty-four mice were randomly and equally divided into four groups: (i) normal diet+saline (Control); (ii) normal diet+casein (ND+CS); (iii) high-fat, high-protein diet+saline (HFPD+SI); (iv) high-fat, high-protein diet+casein (HFPD+CS). After 8weeks, there was a significant increase in body weight for groups HFPD+SI and HFPD+CS and a decrease in group ND+CS compared with the control. The serum levels of tumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10) and lipid profiles were increased markedly in groups ND+CS, HFPD+SI and HFPD+CS compared with the control. A remarkable reduction of serum adiponectin level occurred in group HFPD+CS compared with group ND+CS. Sperm parameters (sperm count, viability and abnormality) were also adversely affected in groups ND+CS and HFPD+SI. Groups ND+CS and HFPD+SI showed severe pathological changes in testicular tissues. Semiquantitative RT-PCR, Western blot and immunohistochemical staining also showed significant reductions in both testicular mRNA and protein levels of steroidogenic acute regulatory (StAR) and cytochrome P450scc (CYP11A1) in groups HFPD+SI and HFPD+CS compared with the control, whereas testicular mRNA and protein levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) in groups HFPD+SI and HFPD+CS significantly increased. The mRNA and protein levels of the StAR and 3β-HSD in group HFPD+CS were both higher than those of in group ND+CS. These results indicated that Kunming male mice with high-fat, high-protein diet and casein injection for 8weeks can be used to establish a diet-induced obesity and chronic systemic inflammation. The sperm parameters in groups ND+CS and HFPD+SI decreased accompanied by pathological changes of testicular tissue. This resultant effect of reduced serum testosterone levels was associated with the overproduction of TNF-α and IL-10 and down-regulation of StAR and CYP11A1. Under the same casein-induced chronic inflammation condition, the mice with high-fat, high-protein diet had better testicular steroidogenesis activity and sperm parameters compared with the mice in normal diet, indicating that the mice with casein-induced inflammatory injury consuming a high-fat, high-protein diet gained weight normally, reduced serum adiponectin level and increased testosterone production by an upregulation of 3β-HSD expression. High-fat, high-protein diet attenuated the negative impact of casein-induced chronic inflammation on testicular steroidogenesis and sperm parameters.

Inflammatory Stress Sensitizes the Liver to Atorvastatin-Induced Injury in ApoE-/- Mice.

Statins, which are revolutionized cholesterol-lowing agents, have been reported to have unfavorable effects on the liver. Inflammatory stress is a susceptibility factor for drug-induced liver injury. This study investigated whether inflammatory stress sensitized the liver to statin-induced toxicity in mice and explored the underlying mechanisms. We used casein injection in ApoE-/- mice to induce inflammatory stress. Half of the mice were orally administered atorvastatin (10mg/kg/d) for 8 weeks. The results showed that casein injection increased the levels of serum pro-inflammatory cytokines (IL-6 and TNFα). Atorvastatin treatment increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in casein injection mice. Moreover, atorvastatin treatment exacerbated hepatic steatosis, inflammation and fibrosis, as well as increased hepatic reactive oxygen species (ROS) and malondialdehyde in casein injection mice. However, above changes were not observed in atorvastatin treated alone mice. The protein expression of liver nuclear factor erythroid 2-related factor 2 (Nrf2) and the mRNA expressions of Nrf2 target genes were increased, together with the enhancement of activities of hepatic catalase and superoxide dismutase in atorvastatin treated alone mice, but these antioxidant responses were lost in mice treated with atorvastatin under inflammatory stress. This study demonstrates that atorvastatin exacerbates the liver injury under inflammatory stress, which may be associated with the loss of adaptive antioxidant response mediated by Nrf2.

Paradoxical effect of rapamycin on inflammatory stress-induced insulin resistance in vitro and in vivo.

Insulin resistance is closely related to inflammatory stress and the mammalian target of rapamycin/S6 kinase (mTOR/S6K) pathway. The present study investigated whether rapamycin, a specific inhibitor of mTOR, ameliorates inflammatory stress-induced insulin resistance in vitro and in vivo. We used tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) stimulation in HepG2 hepatocytes, C2C12 myoblasts and 3T3-L1 adipocytes and casein injection in C57BL/6J mice to induce inflammatory stress. Our results showed that inflammatory stress impairs insulin signaling by reducing the expression of total IRS-1, p-IRS-1 (tyr632), and p-AKT (ser473); it also activates the mTOR/S6K signaling pathway both in vitro and in vivo. In vitro, rapamycin treatment reversed inflammatory cytokine-stimulated IRS-1 serine phosphorylation, increased insulin signaling to AKT and enhanced glucose utilization. In vivo, rapamycin treatment also ameliorated the impaired insulin signaling induced by inflammatory stress, but it induced pancreatic β-cell apoptosis, reduced pancreatic β-cell function and enhanced hepatic gluconeogenesis, thereby resulting in hyperglycemia and glucose intolerance in casein-injected mice. Our results indicate a paradoxical effect of rapamycin on insulin resistance between the in vitro and in vivo environments under inflammatory stress and provide additional insight into the clinical application of rapamycin.

Chronic inflammation aggravates metabolic disorders of hepatic fatty acids in high-fat diet-induced obese mice.

The prevalence of nonalcoholic fatty liver disease (NAFLD) increases with increasing body mass index (BMI). However, approximately 40–50% of obese adults do not develop hepatic steatosis. The level of inflammatory biomarkers is higher in obese subjects with NAFLD compared to BMI-matched subjects without hepatic steatosis. We used a casein injection in high-fat diet (HFD)-fed C57BL/6J mice to induce inflammatory stress. Although mice on a HFD exhibited apparent phenotypes of obesity and hyperlipidemia regardless of exposure to casein injection, only the HFD+Casein mice showed increased hepatic vacuolar degeneration accompanied with elevated inflammatory cytokines in the liver and serum, compared to mice on a normal chow diet. The expression of genes related to hepatic fatty acid synthesis and oxidation were upregulated in the HFD-only mice. The casein injection further increased baseline levels of lipogenic genes and decreased the levels of oxidative genes in HFD-only mice. Inflammatory stress induced both oxidative stress and endoplasmic reticulum stress in HFD-fed mice livers. We conclude that chronic inflammation precedes hepatic steatosis by disrupting the balance between fatty acid synthesis and oxidation in the livers of HFD-fed obese mice. This mechanism may operate in obese individuals with chronic inflammation, thus making them more prone to NAFLD.

Inflammatory stress exacerbates lipid accumulation and podocyte injuries in diabetic nephropathy.

Diabetic nephropathy (DN) is a chronic inflammatory disease that is accompanied by different degrees of lipid disorders. The present study was conducted to determine whether inflammatory stress exacerbates lipid accumulation in podocytes and to investigate its underlying mechanisms in DN using in vitro and in vivo studies. We used IL-1β stimulation in podocytes in vitro and casein injections in db/db mice in vivo to induce inflammatory stress. The plasma levels of serum inflammatory cytokines were determined using an enzyme-linked immunosorbent assay. The renal pathology was evaluated using pathological staining and electron microscopy. Intracellular lipid accumulation was evaluated by Oil Red O staining and a cholesterol quantitative assay. The gene and protein expression levels of extracellular matrix proteins, biomarkers of podocyte injury, and molecules involved in the LDLr pathway were evaluated using immunofluorescence staining, real-time PCR, and western blot analysis. Increased plasma levels of inflammatory cytokines in the casein-injected db/db mice indicated a successful induction of the inflamed DN model. The kidney morphological changes, podocyte injury, and epithelial mesenchymal transition (EMT) were more significant in casein-injected db/db mice. Moreover, inflammation increased the lipid droplet accumulation in the kidneys of db/db mice, which resulted from the increased protein expression levels of LDLr, sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), and SREBP-2 in the kidneys of db/db mice. The in vitro studies further demonstrated that inflammation increased the lipid accumulation in the podocytes and induced podocyte EMT, which were correlated with inflammation-mediated increases in the expression levels of LDLr, SCAP, and SREBP-2, and increased translocation of the SCAP/SREBP-2 complex from the endoplasmic reticulum to the Golgi in the podocytes. Inflammation induced lipid accumulation and the EMT of podocytes through the dysregulation of the LDLr pathway, which contributed to podocyte injury and accelerated the progression of DN.

Inflammatory Stress Exacerbated Mesangial Foam Cell Formation and Renal Injury via Disrupting Cellular Cholesterol Homeostasis.

Inflammation and lipids play significant roles in the progression of chronic kidney disease. This study was designed to investigate whether inflammation disrupts cellular cholesterol homeostasis and causes the lipid nephrotoxicity in vitro and in vivo, and explored its underlying mechanisms. Inflammatory stress was induced by cytokines (interleukin-1β (IL-1β); tumor necrosis factor α (TNF-α)) to human mesangial cells (HMCs) in vitro and by subcutaneous casein injection in C57BL/6J mice in vivo. The data showed that inflammatory stress exacerbated renal cholesterol ester accumulation in vitro and in vivo. Inflammation increased cellular cholesterol uptake and synthesis via upregulating the expression of low-density lipoprotein receptor (LDLr) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoA-R), while it decreased cholesterol efflux via downregulating the expression of liver X receptor alpha and ATP-binding cassette transporter A1. The increased lipid accumulation by inflammatory stress induced reactive oxygen species (ROS) and increased levels of endoplasmic reticulum (ER) stress markers (inositol-requiring protein 1 and activating transcription factor 6) in HMCs and kidneys of C57BL/6J mice. This study implied that inflammation promoted renal lipid accumulation and foam cell formation by disrupting cellular cholesterol homeostasis. Increased intracellular lipids under inflammatory stress caused oxidative stress and ER stress in vitro and in vivo which may contribute to renal injury and progression of chronic kidney disease.

Inflammatory stress increases hepatic CD36 translational efficiency via activation of the mTOR signalling pathway.

Inflammatory stress is an independent risk factor for the development of non-alcoholic fatty liver disease (NAFLD). Although CD36 is known to facilitate long-chain fatty acid uptake and contributes to NAFLD progression, the mechanisms that link inflammatory stress to hepatic CD36 expression and steatosis remain unclear. As the mammalian target of rapamycin (mTOR) signalling pathway is involved in CD36 translational activation, this study was undertaken to investigate whether inflammatory stress enhances hepatic CD36 expression via mTOR signalling pathway and the underlying mechanisms. To induce inflammatory stress, we used tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) stimulation of the human hepatoblastoma HepG2 cells in vitro and casein injection in C57BL/6J mice in vivo. The data showed that inflammatory stress increased hepatic CD36 protein levels but had no effect on mRNA expression. A protein degradation assay revealed that CD36 protein stability was not different between HepG2 cells treated with or without TNF-α or IL-6. A polysomal analysis indicated that CD36 translational efficiency was significantly increased by inflammatory stress. Additionally, inflammatory stress enhanced the phosphorylation of mTOR and its downstream translational regulators including p70S6K, 4E-BP1 and eIF4E. Rapamycin, an mTOR-specific inhibitor, reduced the phosphorylation of mTOR signalling pathway and decreased the CD36 translational efficiency and protein level even under inflammatory stress resulting in the alleviation of inflammatory stress-induced hepatic lipid accumulation. This study demonstrates that the activation of the mTOR signalling pathway increases hepatic CD36 translational efficiency, resulting in increased CD36 protein expression under inflammatory stress.

Inflammatory stress reduces the effectiveness of statins in the kidney by disrupting HMGCoA reductase feedback regulation.

Patients with chronic kidney disease (CKD) are unlikely to gain the same benefit from conventional doses of statins as do patients with cardiovascular disease alone. This study investigated whether inflammation accompanying CKD causes statin resistance. Inflammatory stress was induced by adding cytokines and lipopolysaccharide (LPS) to human mesangial cells (HMCs) in vitro, and in vivo by subcutaneous casein injection in apolipoprotein E, scavenger receptors class A and CD36 triple knockout mice. Inflammatory stress exacerbated cholesterol accumulation and was accompanied in vitro and in vivo by increased HMGCoA reductase (HMGCoA-R) mRNA and protein expression mediated via activation of the sterol regulatory element-binding protein cleavage-activating protein (SCAP)/sterol regulatory element-binding protein 2 pathway. Atorvastatin reduced HMGCoA-R enzymatic activity and intracellular cholesterol synthesis in vitro; however, inflammatory stress weakened these suppressive effects. Atorvastatin at concentrations of 15 µM inhibited HMGCoA-R activity by 50% (IC50) in HMCs, but the same concentration in the presence of interleukin (IL)-1β resulted in only 30% inhibition of HMGCoA-R activity in HMCs. Knocking down SCAP prevented statin resistance induced by IL-1β, and overexpression of SCAP-induced statin resistance even without inflammatory stress. In vivo, the amount of atorvastatin required to lower serum cholesterol and decrease kidney lipid accumulation rose from 2 to 10 mg/kg/day in the presence of inflammatory stress. Inflammatory stress can disrupt HMGCoA-R-mediated cholesterol synthesis resulting in intracellular lipid accumulation and statin resistance.

Establishment of an Inflamed Animal Model of Diabetic Nephropathy

Aims Inflammatory stress plays a crucial role in the progression of diabetic nephropathy (DN). This study aimed to establish a novel inflamed animal model of DN and to evaluate its significance in DN.Methods Nondiabetic db/m mice and diabetic db/db mice were randomly divided into four groups: db/m, db/m+casein, db/db, and db/db+casein for eight weeks. Casein was subcutaneously injected to induce chronic inflammation. Body weight and albumin to creatinine ratio (ACR) in the urine were measured every week. The plasma levels of serum amyloid protein A (SAA) and tumour necrotic factor-α (TNF-α) were determined with the enzyme-linked immunosorbent assay. The morphological changes to the renal pathology and ultra-microstructures were checked by pathological staining and electron microscopy. Immunofluorescent staining and Western blotting were used to determine the protein expression of podocyte-specific molecules and inflammatory cytokines in kidneys.Results ACR, plasma levels of SAA and TNF-α, protein expression of inflammatory cytokines, mesangial expansion, collagen accumulation, and foot process effacement in kidneys of casein-injected db/db mice were significantly increased compared with the db/db mice. Casein injection markedly decreased the protein expression of Wilms' tumor-1 and nephrin in kidneys of db/db mice, which are specific podocyte biomarkers, suggesting that chronic inflammation accelerates podocyte injuries in db/db mice. Interestingly, no obvious urinary protein, inflammatory cytokine expression, or histological changes in the kidneys of casein-injected db/m mice were found compared with the db/m mice.Conclusion An inflamed animal model of DN was successfully established and may provide a useful tool for investigating the pathogenesis of DN under inflammatory stress.

Inflammatory Stress Induces Statin Resistance by Disrupting 3-Hydroxy-3-Methylglutaryl-CoA Reductase Feedback Regulation

The risk of cardiovascular disease is increased by up to 33 to 50× in chronic inflammatory states and convention doses of statins may not provide the same cardiovascular protection as in noninflamed patients. This study investigated whether the increase in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA-R)-mediated cholesterol synthesis observed under inflammatory stress was resistant to the action of statins and if so, whether this was because of interference with the sterol regulatory element binding protein cleavage-activating protein pathway. Inflammatory stress was induced by adding cytokines (interleukin-1β, tumor necrosis factor-α, and interleukin-6) and lipopolysaccharides to vascular smooth muscle cells in vitro and by subcutaneous casein injection in apolipoprotein E/scavenger receptors class A/CD36 triple knockout mice in vivo. Inflammatory stress exacerbated cholesterol ester accumulation and was accompanied in vitro and in vivo by increased HMGCoA-R mRNA and protein expression mediated via activation of the sterol regulatory element binding protein cleavage-activating protein/sterol regulatory element binding protein-2 pathway. Atorvastatin reduced HMGCoA-R enzymatic activity and intracellular cholesterol synthesis in vitro. However, inflammatory stress weakened these suppressive effects. Atorvastatin at concentrations of 16 μmol/L inhibited HMGCoA-R activity by 50% in vascular smooth muscle cells, but the same concentration resulted in only 30% of HMGCoA-R activity in vascular smooth muscle cells in the presence of interleukin-1β. Knocking down sterol regulatory element binding protein cleavage-activating protein prevented statin resistance induced by interleukin-1β, and overexpression of sterol regulatory element binding protein cleavage-activating protein induced statin resistance even without inflammatory stress. In vivo, the amount of atorvastatin required to lower serum cholesterol and decrease aortic lipid accumulation rose from 2 to 10 mg/kg per day in the presence of inflammatory stress. Increased cholesterol synthesis mediated by HMGCoA-R under inflammatory stress may be one of the mechanisms for intracellular lipid accumulation and statin resistance.

Chronic inflammation exacerbates glucose metabolism disorders in C57BL/6J mice fed with high-fat diet

Inflammatory stress is closely related to metabolic disease and insulin resistance. The precise cellular mechanism linking obesity and diabetes is largely unknown, but about 14-20% of obese individuals develop diabetes. In this study, we investigated whether chronic inflammation exacerbated glucose metabolism disorder by impairing β cell function in high-fat diet (HFD)-fed C57BL/6J mice. We used s.c. casein injection to induce chronic inflammation in HFD-fed C57BL/6J mice; 14 weeks on a HFD resulted in weight gain, hyperlipidemia, and low insulin sensitivity in these mice which nevertheless had normal blood glucose and serum inflammatory cytokines levels. Casein injection in the background of HFD elevated serum tumor necrosis factor α (TNFα) and serum amyloid A levels and increased TNFα and MCP1 expression in the adipose tissue, liver, and muscle of HFD-fed mice. Chronic inflammation induced by casein injection further decreased insulin sensitivity and insulin signaling, resulting in insulin deficiency and hyperglycemia in these mice. Islet mass and insulin content were markedly increased in HFD mice. However, in contrast with HFD-fed alone, chronic inflammation in HFD-fed mice decreased both islet mass and insulin content, reduced the genetic expression of insulin synthesis and secretion, and increased β cell apoptosis. We conclude that chronic inflammation exacerbated glucose metabolism disorders by impairing β cell function in HFD-fed C57BL/6J mice, suggesting that this mechanism may operate in obese individuals with chronic inflammation, making them prone to hyperglycemia.

Inflammatory Stress Exacerbates the Progression of Cardiac Fibrosis in High-Fat-Fed Apolipoprotein E Knockout Mice via Endothelial-Mesenchymal Transition

Background Chronic inflammation plays a crucial role in the progression of cardiac fibrosis. This study investigated whether inflammation exacerbated the progression of cardiac fibrosis in high-fat-fed apolipoprotein E knockout (ApoE KO) mice via endothelial-mesenchymal transition (EndMT).Methods Twenty-four male ApoE KO mice were divided into normal chow diet (Control), high-fat diet (HFD), or high-fat diet plus 10% casein injection (inflamed) groups for 8 weeks. The body weight of ApoE KO mice was measured at each week. The lipid profile and serum amyloid A (SAA) levels were examined using clinical biochemistry and enzyme-linked immunosorbent assays, respectively. Cardiac lipid and collagen accumulation was visualised with haematoxylin-eosin (HE) and Masson's trichrome staining. EndMT-related molecule expression was examined by immunohistochemistry and Western blotting.Results SAA levels were increased in the inflamed group compared with the HFD and control groups, suggesting that inflammation was successfully induced. There were no differences in body weight among three groups at each week. Interestingly, inflammation significantly reduced serum total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels compared with the HFD mice. However, both foam cell formation in cardiac blood vessels and cardiac collagen deposition were increased in the inflamed group, as demonstrated by HE and Masson trichrome staining. Furthermore, inflammation reduced protein expression of CD31 and increased protein expression of alpha-smooth muscle actin (α-SMA) and collagen I, which contribute to cardiac EndMT.Conclusions Inflammatory stress exacerbates the progression of cardiac fibrosis in high-fat-fed ApoE KO mice via EndMT, suggesting that hyperlipidaemia and inflammation act synergistically to redistribute plasma lipids to cardiac tissues and accelerate the progression of cardiac fibrosis.

Rosiglitazone attenuates casein-induced hepatic endoplasmic reticulum stress in Sprague-Dawley rats: a novel model of endoplasmic reticulum stress

The proteins found in cow milk have been reported to cause systemic inflammation. Endoplasmic reticulum (ER) stress is known to be involved in the development of several metabolic disorders including insulin resistance and non-alcoholic fatty liver disease. However, the effect of thiazolidinediones (TZDs) on ER stress is still controversial. This is why we want to investigate in this study whether casein, which is the major protein in cow's milk, induces ER stress in the liver and whether rosiglitazone can attenuate these changes. Nine-week-old male Sprague-Dawley (SD) rats were separated into three groups: (1) vehicle treated; (2) daily subcutaneous injections of 1 mL 10% casein; (3) daily subcutaneous injection of 1 mL 10% casein and rosiglitazone 4 mg/[kg d]. After 6 weeks, body weight, food intake, glucose and lipid parameters, and serum AST/ALT levels were measured after an overnight fast. Real time RT-PCR and immunohistochemical staining for various ER stress markers were performed, and a TUNEL analysis was also performed. After 6 weeks, casein injection induced weight reduction, systemic inflammation, and hepatic dysfunction in SD rats. Casein injection increased both the gene and protein expression of ER stress markers in the liver and also caused hepatocyte apoptosis. Rosiglitazone treatment attenuated casein-induced systemic inflammation, ER stress, deteriorated liver function, and increased apoptosis. In conclusion, our results may provide further insight into the effects of casein on chronic inflammatory diseases, and to have a better understanding of the mechanism of the anti-inflammatory properties of rosiglitazone regardless of its hypoglycemic effect.

Inflammatory stress exacerbates hepatic cholesterol accumulation via disrupting cellular cholesterol export

Both inflammation and cholesterol accumulation play important roles in the development of non-alcoholic fatty liver disease. This study was undertaken to investigate whether inflammation aggravated cholesterol accumulation via disrupting hepatic cholesterol export and we explored the underlying mechanisms. We used casein injection in C57BL/6J mice, and tumor necrosis factor alpha (TNF-α) stimulation in human hepatoblastoma cell line (HepG2) cells to induce inflammation. Intracellular cholesterol level was examined by Oil Red O staining and quantitative analysis. Bile acid level was quantified by colorimetric analysis. (3)[H] cholesterol assay by scintillation counting was performed to evaluate the cholesterol efflux. The mRNA and protein expression was examined by real-time polymerase chain reaction and Western blot. Inflammation increased cholesterol accumulation in livers of C57BL/6J mice and in HepG2 cells. High-fat diet in mice and low-density lipoprotein (LDL) loading in HepG2 cells increased bile acid synthesis and cholesterol efflux, enhanced the mRNA and protein expression of liver X receptor α (LXRα), peroxisome proliferator-activated receptors (PPARα, γ), cholesterol 7α-hydroxylase (CYP7A1) and ATP-binding cassette transporter A1 (ABCA1). However, inflammation reduced bile acid synthesis and cholesterol efflux even in high-fat-diet-fed mice and HepG2 cells in the presence of LDL loading. The enhanced effects of these genes and proteins expression due to high-fat diet and LDL loading were inhibited by inflammation both in vivo and in vitro. Inflammation disrupted PPAR-LXR-CYP7A1/ABCA1-mediated bile acid synthesis and cholesterol efflux resulting in exacerbated cholesterol accumulation in livers of C57BL/6J mice and HepG2 cells.

Inflammatory stress exacerbates lipid-mediated renal injury in ApoE/CD36/SRA triple knockout mice

Both lipids and inflammation play important roles in the progression of kidney disease. This study was designed to investigate whether inflammation exacerbates lipid accumulation via LDL receptors (LDLr), thereby causing renal injury in C57BL/6J mice, apolipoprotein E (ApoE) knockout (KO) mice, and ApoE/CD36/scavenger receptor A triple KO mice. The mice were given a subcutaneous casein injection to induce inflammatory stress. After 14 wk, terminal blood samples were taken for renal function, lipid profiles, amyloid A (SAA), and IL-6 assays. Lipid accumulation in kidneys was visualized by oil red O staining. Fibrogenic molecule expression in kidneys was examined. There was a significant increase in serum SAA and IL-6 in the all casein-injected mice compared with respective controls. Casein injection reduced serum total cholesterol, LDL cholesterol, and HDL cholesterol and caused lipid accumulation in kidneys from three types of mice. The expression of LDLr and its regulatory proteins sterol-responsive element-binding protein (SREBP) 2 and SREBP cleavage-activating protein (SCAP) were upregulated in inflamed mice compared with controls. Casein injection induced renal fibrosis accompanied by increased expression of fibrogenic molecules in the triple KO mice. These data imply that inflammation exacerbates lipid accumulation in the kidney by diverting lipid from the plasma to the kidney via the SCAP-SREBP2-LDLr pathway and causing renal injury. Low blood cholesterol levels, resulting from inflammation, may be associated with high risk for chronic renal fibrosis.

Inflammatory stress exacerbates ectopic lipid deposition in C57BL/6J mice

BackgroundChronic systemic inflammation and abnormal free fatty acid metabolism are closely related to ectopic lipid deposition. In this study, we investigate if inflammation tissue-specifically disrupts lipogenesis and lipolysis in nonadipose tissues and adipose tissue, resulting in ectopic lipid deposition in C57BL/6J mice.MethodsWe used casein injection in C57BL/6J mice to induce a chronic systemic inflammatory stress in vivo. Serum was analyzed for free fatty acid and cytokines. Insulin sensitivities were evaluated by glucose and insulin tolerance tests. Liver, muscle, adipose tissues were taken for lipid analysis. Real-time polymerase chain reaction and western blotting were used to examine the gene and protein expression of molecules involved in adipogenesis and lipolysis in tissues.ResultsCasein injection elevated serum levels of IL-6 and SAA in mice, which are associated with increased lipid accumulation in liver and muscle, suggesting that chronic systemic inflammation induces ectopic lipid deposition in nonadipose tissues. The inflammatory stress upregulated mRNA and protein expression of sterol regulatory element binding protein 1, fatty acid synthase, and acetyl CoA carboxylase alpha, while inhibited these molecules expression in adipose. Interestingly, in the same experimental setting, inflammation increased triglyceride lipase and hormone-sensitive lipase expression in white adipose tissue. Inflammation also induced insulin resistance and increased serum free fatty acid levels in C57BL/6J mice.ConclusionsChronic systemic inflammation increased lipogenesis in nonadipose tissues and lipolysis in white adipose tissue, resulting in ectopic lipid deposition in nonadipose tissues. This disturbed free fatty acid homeostasis and caused insulin resistance in C57BL/6J mice.

Inflammatory stress exacerbates hepatic cholesterol accumulation via increasing cholesterol uptake and de novo synthesis

Cholesterol accumulation plays an important role in the progression of non-alcoholic fatty liver disease. We have demonstrated that inflammation aggravated cholesterol accumulation, causing tissue injury in the vessel and kidney. This study was undertaken to investigate whether inflammatory stress exacerbates hepatic cholesterol accumulation and we explored the underlying mechanisms. We used casein injection in C57BL/6J mice, interleukin-1β and interleukin-6 stimulation in human hepatoblastoma cell line (HepG2) cells to induce inflammatory stress. Oil Red O staining and intracellular cholesterol assay were used to quantify cellular cholesterol levels. Real-time reverse transcription polymerase chain reaction and Western blot were used to measure messenger RNA (mRNA) and protein expression of target genes. HMGCoA reductase (HMGCoA-r) enzymatic activity and cellular cholesterol synthesis were measured by radioactive methods. We demonstrated that inflammatory stress increased hepatic cholesterol accumulation and enhanced sterol regulatory element binding protein 2 (SREBP2), low-density lipoprotein receptor (LDLr) and HMGCoA-r mRNA and protein expression in livers of C57BL/6J mice and in HepG2 cells. A high-fat diet in mice or LDL loading in HepG2 cells inhibited mRNA and protein expression of these genes. However, the suppressive effect was overridden by inflammatory stress both in vivo and in vitro. Inflammatory stress increased HMGCoA-r enzymatic activity and cellular cholesterol synthesis in HepG2 cells in the absence or presence of LDL loading. Inflammatory stress disrupted hepatic SREBP2-mediated low-density lipoprotein receptor and HMGCoA-r feedback regulation resulting in exacerbated cholesterol accumulation in livers of C57BL/6J mice and HepG2 cells.

Modulation of mammary gland development in pre-pubertal mice as affected by soya and milk protein supplements

The objective of the present study was to determine the effects of soya and whey milk protein, α-lactalbumin (α-LA), on mammary gland morphology and the structural support of the gland, in pre-pubertal mice after 7 d of treatment. In Expt 1, weaned (day 21) CD1 mice were given one of the four treatments, three included dietary supplements: (1) control diet, casein, (2) soya, (3) α-LA and (4) subcutaneous injection of 2·5 μg oestradiol benzoate in 20 μl maize oil and fed the control diet. All diets were isoenergetic with equal protein concentrations. All groups that were not treated with oestradiol received the vehicle. Whole-mount analyses were performed to determine longitudinal ductal growth and terminal end bud development. DNA was extracted from the gland and assessed by spectrophotometry (260/280 nm). Tissue extracts for extracellular matrix (ECM) proteins, matrix metalloproteinase-2 (MMP(2)), tissue inhibitor of MMP(2) (TIMP(2)), and serum oestradiol and mammary tissue epidermal growth factors (EGF) were measured by immunoassays. Expt 2 utilised the Her2/neu transgenic strain, with the same protocols. Statistical significance was determined by one-way ANOVA. From Expt 1 and 2, soya and α-LA significantly increased ductal elongation when compared with the oestrogen and control groups. These results were corroborated by data on total DNA and the ratio of MMP(2):TIMP(2). The ratio of MMP(2):TIMP(2) was affected by α-LA. Serum oestradiol was decreased only in the oestradiol-treated groups in both experiments. Soya is known to be oestrogenic and can act on epithelia directly. The mechanism by which α-LA affects glandular development is by modulating the ECM or by promoting the synthesis/activity of EGF.