Sphingosine-1-phosphate receptor 2 attenuates influenza A virus-induced viral pneumonia through PI3K/AKT/eNOS pathway

AIM: To investigate the effects of sphingosine-1-phosphate receptor 2 (S1PR2) on influenza A virus-induced viral pneumonia.METHODS: The animal model of influenza A virus pneumonia was established by infecting wild-type C57BL/6 mice and S1pr2^-/- mice with influenza virus subtype FM1 mouse lung adaptable strain through nose drops. The pathological changes of the lung tissues of wild-type mice (model group), JTE-013 (S1PR2 effective antagonist)-challenged mice and S1pr2^-/- mice were observed, and the protein concentration, total cell number, and interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) levels were determined in the bronchoalveolar lavage fluid (BALF) at 4 d and 6 d after virus infection. The phosphorylation levels of AKT and eNOS in the lung tissues were determined by Western blot. RESULTS: Compared with the wild-type mice of control group, the influenza A virus pneumonia in JTE treatment group and S1pr2^-/- mice were more serious, and the protein concentration, total cell number and inflammatory cytokines in the BALF were remarkably increased. Moreover, the phosphorylation levels of AKT and eNOS, the downstream targets of PI3K, were significantly increased (P<0.01). CONCLUSION: S1PR2 mediates PI3K/AKT/eNOS signaling transduction pathway to regulate NO generation, and inhibit vascular permeability and inflammatory cytokine release, thus attenuating the viral pneumonia induced by influenza A virus.     

Mutation of Npc1 gene causes abnormal lipid metabolism to induce apoptosis of renal cells and promote fibrosis

AIM: To investigate the dysfunction of renal cell and tissue in Npc1 mutant mice, in order to provide support for the treatment of Niemann-Pick disease type C1 (NPC1) patients.METHODS: The kidneys of wild-type (Npc1^+/+) and Npc1 mutant (Npc1^-/-) mice on postnatal day 60 were isolated. HE staining was performed to examine the morphological changes of the renal tissues. Oil red O staining was used to examine the lipid deposition in the renal tissues. The apoptosis of the renal cells was detected by TUNEL staining. The expression of apoptosis-related proteins in the renal tissue was determined by Western blot, and immunofluorescence was performed to examine the expression of α-smooth muscle actin (α-SMA) and vimentin in the renal tissues. RESULTS: Compared with Npc1^+/+ mice, the morphological observation showed obvious vacuoles and no lipid deposition in the renal tissue of Npc1^-/- mice. Subsequently, TUNEL staining showed significant increase in the apoptotic cells in the renal tissue of Npc1^-/- mice (P<0.01), and the expression levels of Bax and Bad were up-regulated in the renal tissues of Npc1^-/- mice (P<0.01), but Bcl-2 was down-regulated (P<0.05). Furthermore, the expression of α-SMA and vimentin was significantly up-regulated in the renal tissues of Npc1^-/- mice (P<0.01). CONCLUSION: Npc1 gene mutation causes abnormal lipid metabolism in the renal cells, which induces the apoptosis of renal cells and promotes the fibrosis of renal tissue.     

Sphingosine-1-phosphate receptor-2 attenuates lipopolysaccharide-induced acute lung injury

AIM: To investigate the effects and mechanisms of sphingosine-1-phosphate receptor-2 (S1P2R)on lipopolysaccharide (LPS)-induced acute lung injury (ALI). METHODS: ALI model was induced by intratracheal administration of LPS in both wild-type mice and S1P2R -deficient mice. The pathological changes in the lung tissues were observed, and the protein concentration, total cell number, neutrophil ratio, TNF-α level and IL-6 level were determined in the bronchoalveolar lavage fluid (BALF) 24 h after LPS injection. In order to investigate the mechanisms of S1P2R in LPS-induced ALI, 10 min before LPS injection, both wild-type mice and S1P2R -deficient mice were injected with nitric oxide synthase inhibitor by tail vein injection, the pathological changes of the lung tissues were observed, and the protein concentration and total cell number in BALF were determined 12 h after LPS injection. RESULTS: Compared with wild-type mice, S1P2R -deficient mice showed more severe LPS-induced ALI, and the protein concentration, neutrophils and inflammatory cytokines in BALF were significantly increased in S1P2R -deficient mice. Administration of nitric oxide synthase inhibitor N^ω-L-nitro-arginine methyl ester protected S1P2R -deficient mice from aggravation of ALI. CONCLUSION: S1P2R mediates the protection from LPS-induced ALI possibly through inhibiting nitric oxide synthase.     

Renal function and pathological changes in Niemann-Pick disease type C1 mice

AIM: To investigate the renal function and pathological changes in Npc1 mutant (Npc1^-/-) mice. METHODS: Different genotypes of Niemann-Pick disease type C1 (Npc1) mice were identified by PCR. Subsequently, the renal function of Npc1^-/- and Npc1^+/+ mice at postnatal day 60 (P60) was evaluated by measuring the activity and content of important indicators in the serum including ALT, AST, LDH, urea, UA and Cr. Furthermore, β-galactosidase staining and Masson staining were performed to examine the aging and fibrosis of the renal tissues, respectively. RESULTS: Compared with the Npc1^+/+ mice, the body weight and kidney weight had a significant reduction (P<0.01) in the Npc1^-/- mice. The results of hepatic and renal functions showed that the activities of ALT, AST and LDH, and contents of urea, UA and Cr had marked increases (P<0.05) in the Npc1^-/-mice. Moreover, the results of senescence-associated β-galactosidase staining in the renal tissues demonstrated accelerated aging in the Npc1^-/- mice (P<0.01), and these results were confirmed by Masson staining, which clearly showed the formation of collagen fibers (P<0.01).CONCLUSION: Mutation of the Npc1 gene results in abnormal lipid metabolism, which accelerates kidney senescence by promoting fibrosis in the renal tissue and subsequently causes reduction in renal function.     

Effects of p38 MAPK on SETD4 expression and location in p38 +/+ and p38 -/- cells treated with sodium arsenite

AIM: To investigate the expression and location of SET domain-containing 4(SETD4) protein in p38 ^+/+ and p38 ^-/- cells treated with sodium arsenite(NaAsO₂). METHODS: The expression and location of SETD4 were detected in different cells with or without NaAsO₂ treatment by Western blotting and immunofluorescence technique. RESULTS: The expression of SETD4 was detectable in both murine- and human-derived cells. Its distribution was found to be located in the whole cell, mainly in the cytoplasm. Further investigation also suggested that the protein expression of SETD4 was reduced in both p38 ^+/+ and p38 ^-/- cells 6 h after NaAsO₂ treatment. Moreover, SETD4 protein was translocated from cytoplasm to nucleus in p38 ^+/+ cells treated with NaAsO₂, which was unobvious in p38 ^-/- cells. CONCLUSION: SETD4 protein is expressed in various cells derived from different species and tissues, and it is mainly located in cytoplasm. NaAsO₂ treatment influences the expression of SETD4, and induces the translocation of SETD4 protein to nucleus, which might be involved in the p38 MAPK signal pathway.     

Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

Role of P-selectin in intestinal tumorigenesis in ApcMin/+ mice

AIM: To investigate the role of P-selectin in intestinal tumorigenesis in Apc^Min/+ mice (C57BL/6J-Apc^Min/J). METHODS: Female P-selectin knockout mice (B6.129S7-Selp^tm1Bay /J, that was P-selectin ^-/-) were mated with male Apc^Min/+ mice. The offspring was genotyped for Min ^+/- and P-selectin null mutantions, which were Apc^Min/+ P-selectin ^-/- mice. The tumor number and gross tumor volume in the small and large intestines of the Apc^Min/+ P-selectin ^-/- mice and Apc^Min/+ mice were determined. RESULTS: P-selectin deficiency in Apc^Min/+ mice resulted in significant decreases in the tumor number and gross tumor volume in the small intestine and total intestine. CONCLUSION: Deletion of P-selectin significantly inhibits the tumorigenesis in mouse intestines. In addition, the results also suggest that P-selectin may be a marker for colon cancer.     

Effect of interleukin-10 on attenuating endotoxin-induced acute lung injury

AIM: To study the role of polymorphonuclear neutrophile(PMN) in lipopolysaccharide (LPS)- induced acute lung injury (ALI) and the protective effect of interleukin -10(IL-10) on ALI. METHODS: LPS alone (100μg) or LPS+ IL-10 (l ug) was instilled intratracheally into rats. PMN numbers, protein content and malondialdehyde (MDA) content in bronchoalveolar lavage fluid (BALF) were measured. Histological change of lung was also observed. RESULTS: LPS increased significantly PMN numbers, protein content and MDA content in BALF. Histological finding shows PMN accumulation in lung. IL - 10+LPS reduced remarkably PMN numbers ,pro- tein content and MDA content in BALF than those caused by LPS. PMN decreasing was also identified by light microscopy. CONCLUSION: LPS instilled intratracheally causes PMN accumulation in lung and ALI, while IL - 10 could alleviate ALI through reducing PMN accumulation.     

Bone marrow-derived mesenchymal stem cell-conditioned medium atte-nuates LPS-induced acute lung injury in mice

AIM:To explore the effects of bone marrow-derived mesenchymal stem cells-conditioned medium (MSCs CdM) on lipopolysaccharide (LPS)-induced acute lung injury. METHODS:Lung injury was induced in mice by intraperitoneal injection of LPS. The mice were given a tail vein injection of MSCs CdM or normal saline 1 h after LPS administration. The mice were killed by an intraperitoneal injection of pentobarbital 6 h after LPS injection for either bronchoalveolar lavage fluid (BALF) and serum collection or lung histological analysis. RESULTS:Compared with control group, the BALF levels of protein, interleukin-10 (IL-10) and keratinocyte growth factor (KGF), the serum levels of tumor necrosis factor α (TNF-α) and IL-6, and the myeloperoxidase (MOP) activity in the lung tissues were significantly higher in LPS group, and severe pathological damages in the lung tissues were also observed. Treatment with MSCs CdM significantly reduced the BALF prtein level, the seum TNF-α and IL-6　levels and the lung MPO activity, and attenuated the lung pathological damages, but further increased the levels of IL-10 and KGF in the BALF. CONCLUSION:Treatment with MSCs CdM attenuates the lung injuries induced by LPS, which may be via regulating the expression of TNF-α, IL-6, IL-10 and KGF.     

Effects of butylphthalide on atherosclerosis lesion and VCAM-1 expression in aortic wall of ApoE-/- mice

AIM: To observe the protective effects of butylphthalide on atherosclerosis lesion and vascular cell adhesion molecular-1 (VCAM-1) expression in the aortic wall of ApoE^-/- mice, and to explore the possible mechanism underlying these beneficial effects.METHODS: Male ApoE^-/- mice at 6 weeks of age (n=90) were randomly divided into 3 groups. Thirty ApoE^-/- mice fed with high-fat diet and treated with saline simultaneously were defined as model group. Thirty ApoE^-/- mice fed with high-fat diet and treated with butylphthalide (100 and 200 mg·kg^-1·d^-1) were defined as treatment groups. Thirty wild-type C57BL/6J mice treated with saline were defined as control group. Fifteen mice in each group were sacrificed both at the ages of 18 and 30 weeks. The body weight, food intake and water intake were monitored weekly through the experiment. The lipid profiles were determined both at 18 and 30 weeks of age. Aortic roots were stained with hematoxylin and eosin for pathological examination. Serum ox-LDL, CRP, TNF-α and IL-6 were examined by ELISA. The expression of VCAM-1 at mRNA and protein levels was determinate by real-time PCR and Western blot in the thoracic aortas. RESULTS: Compared with control group, at 18 and 30 weeks of age, the body weight, serum lipid profiles and inflammatory factors were increased, while the atherosclerotic plaques were raised. The mRNA and protein levels of VCAM-1 were up-regulated. However, serum lipid levels in butylphthalide treatment groups (both at doses of 100 and 200 mg·kg^-1·d^-1) were decreased significantly. Serum ox-LDL, CRP, TNF-α and IL-6 were also decreased by butylphthalide treatment. Furthermore, atherosclerotic plaque areas in the aortic roots were reduced by butylphthalide treatment. In addition, the expression of VCAM-1 at mRNA and protein levels in the thoracic aortas was down-regulated by butylphthalide treatment.CONCLUSION: Butylphthalide delays the occurrence of high-fat diet-induced atherosclerosis and down-regulates the expression of VCAM-1 in the ApoE^-/- mice, which may be due to its alleviative effects on hyperlipidemia and inflammation.     

RXRα agonist bexarotene inhibits atherosclerosis in ApoE-/- mice by regulating TGF-β1/Smad2 signaling pathway

AIM To observe the effect of retinoid X receptor α （RXRα） agonist bexarotene （Bex） on the proliferation of transforming growth factor β1 （TGF-β1）-induced vascular smooth muscle cells （VSMCs） and atherosclerosis in apolipoprotein E knockout （ApoE^-/-） mice， and to explore the underlying mechanism. METHODS Ten C57BL/6 mice were selected as normal control group， and 30 ApoE^-/- mice were randomly divided into 3 groups： ApoE^-/- group， ApoE^-/-+Bex5 （5 mg·kg^-1·d^-1 Bex） group and ApoE^-/-+Bex10 （10 mg·kg^-1·d^-1 Bex） group. Bex was intragastrically given once a day for 8 weeks. The levels of triglyceride （TG） and total cholesterol （TC） were determined by oxidase method， and select masking method was used to determine serum levels of low-density lipoprotein cholesterol （LDL-C） and high-density lipoprotein cholesterol （HDL-C）. The protein levels of TGF-β1， p-Smad2 and Smad2 were determined by Western blot. HE staining was used to observe the intima of the thoracic aorta. The VSMCs were cultured with tissue patch method， and the proliferation of VSMCs was measured by BrdU incorporation method. RESULTS The serum levels of TG， TC and LDL-C， and the expression of TGF-β1 and p-Smad2 in thoracic aorta in ApoE^-/- group were significantly higher than those in C57BL/6 group （P<0.01）. Bex increased p-Smad2 protein level in thoracic aorta in a dose-dependent manner， inhibited the intimal plaque formation and vascular medial proliferation， and decreased the plaque area in ApoE^-/- mice （P<0.01）. No significant difference in serum levels of TG， TC， HDL-C and LDL-C， and TGF-β1 and Smad2 expression in thoracic aorta among ApoE^-/- group， ApoE^-/-+Bex5 group and ApoE^-/-+Bex10 group was observed. TGF-β1 （0.1~10 μg/L） promoted the proliferation of VSMCs， while Bex （10^-9~10^-7 mol/L） inhibited TGF-β1 （5 μg/L）-induced proliferation of VSMCs in a concentration-dependent manner. Bex （10^-7 mol/L） synergistically promoted the protein level of p-Smad2 in VSMCs induced by TGF-β1 （P<0.01）， but inhibited TGF-β1-induced nuclear translocation of p-Smad2. CONCLUSION RXRα agonist Bex inhibits the formation of atherosclerosis in ApoE^-/- mice， and its mechanism may be related to the regulation of TGF-β1/Smad2 pathway.     

Protective effect of recombinant human serum albumin-thioredoxin fusion protein on mice with acute lung injury induced by influenza virus

AIM To investigate the protective effect of recombinant human serum albumin (HSA)-thioredox?in (Trx) fusion protein (HSA-Trx) on mice with acute lung injury (ALI) induced by influenza virus infection. METH?ODS: The recombinant HAS-Trx fusion protein was generated by Pichia pastoris expression system. ICR mice were used to establish the animal model of ALI induced by PR8 (H1N1) influenza virus, and the experimental mice were divided into healthy control group, ALI group, ALI+Trx group and ALI+HSA-Trx group, with 10 mice in each group. The bronchoalveolar lavage fluid (BALF) in each group was collected, the total number of cells and the number of alveolar neutrophils were determined, the protein concentration was measured by Coomassie brilliant blue solution method, and the interferon-γ (IFN-γ) content in BALF was detected by ELISA. The lung tissues were collected for hematoxylin and eosin staining. The inducible nitric oxide synthase (iNOS), 8-hydroxydeoxyguanosine (8-OHdG) and 3-nitrotyrosine (NO₂-Tyr) in lung tissues were detected by immunofluorescence method. Peroxide concentration in plasma was evaluated using a CR2000RC analyzer. RESULTS HSA-Trx treatment significantly reduced the total number of cells, neutrophils and total protein in BALF of ALI mice (P<0.05), and decreased the levels of 8-OHdG, NO₂-Tyr in lung tissue and peroxide in plasma (P<0.05). However, it has no significant inhibitory effect on iNOS and IFN-γ expression (P>0.05). CONCLUSION HSA-Trx inhibits inflammatory response and excessive production of nitric oxide in the lung, thus protecting influ?enza virus-induced ALI mice.     

Sodium nitrite reduces lipopolysaccharide-induced acute lung injury in mice

AIM: To investigate the effect of sodium nitrite (SN) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and its underlying mechanism in mice. METHODS: All male Institute of Cancer Research (ICR) mice were randomly divided into five groups: Control group; LPS group; and SN 4.8 nmol/L, SN 48 nmol/L, SN 480 nmol/L (ip) groups. Lung wet weight/dry weight (W/D) ratio and permeability were detected. Neutrophil infiltration in bronchoalveolar lavage fluid (BALF) was measured by cel1 counting and morphological changes in lung tissues were assayed by hematoxylin-eosin staining. The 1evels of interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) in lung were detected. Nitric oxide (NO) level and nitric oxide synthase (NOS) activity in lung were measured according to the specification. RESULTS: Compared to lung in LPS-induced ALI mice, at doses of 4.8 nmol/L and 48 nmol/L, not 480 nmol/L, SN markedly decreased the lung W/D ratio, total leukocyte number and neutrophil percentage in the BALF, lung permeability, and TNF-α/IL-10 ratio, in lung. SN at dose of 480 nmol/L markedly increased the lung NO level compared to control group. In addition, SN decreased the total NOS and inducible NOS (iNOS) activities compared to LPS-induced ALI mice. CONCLUSION: These results indicate that the protective effect of SN against LPS-induced ALI in mice is associated with the low dose SN-induced NO, as well as the subsequent decrease in iNOS activity and TNF-α/IL-10 ratio.     

Propofol inhibits LPS-induced inflammatory response in microglia via TRPV1 ion channels

AIM:To investigate the effects of propofol (P) on the inflammatory response of microglia induced by lipopolysaccharide (LPS) and the mechanisms. METHODS:Mouse microglia BV2 cells were treated with LPS at 100 μg/L to establish a neuroinflammatory injury model. The BV2 cells were divided into 4 groups:control group (C group), model group (L group), L+P group and LPS+AMG517 group (L+A group). The level of tumor necrosis factor-α (TNF-α) in the cell culture supernatant was measured by ELISA. The mRNA expression of transient receptor potential cation channel subfamily V member 1 (TRPV1) was detected by real-time PCR. The protein levels of TRPV1, TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and phosphorylated calcium/calmodulin-dependent protein kinase Ⅱ (p-CaMKⅡ) were determined by Western blot. The content of free Ca²⁺ in the microglia BV2 cells was detected by Fluo-3 AM assay. RESULTS:Compared with C group, the level of TNF-α was significantly increased in L group (P<0.01), but that in P group was not changed. Compared with L group, the level of TNF-α was significantly lower than that in L+P group within 4 h (P<0.01). Compared with C group, the mRNA expression of TRPV1 was significantly increased in L group (P<0.01). Compared with L group, the mRNA expression of TRPV1 was significantly down-regulated in L+P group (P<0.01).Compared with L group, the protein levels of TNF-α, IL-1β, IL-6 and p-CaMKⅡ and intracellular Ca²⁺ concentration were significantly lower than those in L+P group and L+A group (P<0.01). CONCLUSION:Propofol inhibits the inflammatory response of microglia by reducing the expression of TNF-α, IL-1 and IL-6, which may be related to the down-regulation of TRPV1 and p-CaMKⅡ and the reduction of intracellular Ca²⁺ concentration.     

The cytotoxicity of nitric oxide induced by inflammatory cytokine in combination with LPS in endothelial cells

AIM: To explore the mechanism underlying inducible nitric oxide (NO) caused injury of endothelial cells during inflammation. METHODS:The activity of iso-enzymes of NO synthase (NOS), NO level and iNOS expression were examined using NADPH method, Griess reaction and RT-PCR, respectively. Furthermore, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content were also measured. RESULTS:Co-administration of cytokines (TNF-α 5×10⁵ U/L, IL-1β 2×10⁵ U/L, INF-γ 2×10⁵ U/L) and LPS (10 mg/L) caused an obvious increase in NOS activity, NO levels (about two-fold) and a significant injury of the cells. At the same time, a significant increase in iNOS mRNA was also detected. Wheareas, treatment of the cells separately with cytokines or LPS for 24 h had no significant effect on NOS activity and NO level in cell lysates, however, it caused a significant increase in LDH release and MDA content. Also, the effect of cytokines and LPS on cell viability was concentration-and time-dependent. L-NMMA, a inhibitor of NOS, can suppress inducible NO production and protect cells against NO induced injury. CONCLUSION:Co-administration of cytokines (TNF-α, IL-1β and INF-γ) and LPS significant activated iNOS and NO production which, in turn, induced oxidative reaction in endothelial cells.     

Cytokine levels and the discrepancy of dexamethasone intervening in ALI rats induced by hydrochloric acid and lipopolysaccharide

AIM: To explore the cytokines level and the discrepancy of reaction to dexamethasone (Dex) in ALI rats induced by hydrochloric acid (HCl) and lipopolysaccharide (LPS). METHODS: Ninety-six SD rats were divided into six groups at random (n=16 in each group): NS group, HCl group, LPS group, NS+Dex group, HCl+Dex group and LPS+Dex group. Every group was divided into two subgroups: the bronchoalveolar lavage (BAL) subgroup and no bronchoalveolar lavage (NBAL) subgroup. The total leukocytes, PMN%, macrophage%, lymphocyte%, total protein in BALF and the wet/dry of the lung weight were measured. The concentrations of TNF-α, IL-1β, IL-4 and IL-10 in serum and BALF in every group were compared. RESULTS: (1) In the groups of LPS and HCl, the total leukocytes, PMN numbers, the protein concentration in the BALF and W/D were higher than those in control group (P<0.05). Compared to LPS groups, the percentage of macrophage increased in LPS+Dex group (P<0.05). (2) In serum and BALF of both LPS group and HCl group, the concentrations of TNF-α, IL-4 and IL-10 were higher than those in the control (P<0.01). The content of IL-1β in serum of all the groups was undetected. Compared to LPS groups, the concentrations of TNF-α and that of IL-1β decreased in LPS+Dex group (P<0.05). The concentration of IL-10 in LPS+Dex group was higher than that in LPS group (P<0.01). CONCLUSION: The permeability and the inflammatory cytokines in these two models were not consistent. Glucocorticoids play an effective role for resisting ALI induced by LPS but not HCl.     

Protective role of endogenous hydrogen sulfide in cholecystokinin octapeptid against acute lung injury induced by lipopolysaccharide

AIM: To explore the role of endogenous hydrogen sulfide (H₂S) in the mechanism of cholecystokinin octapeptide (CCK-8) to alleviate acute lung injury (ALI) induced by lipopolysaccharide (LPS). METHODS: Eighty-four Sprague-Dawley rats were randomly divided into seven groups: control, LPS (instilled intratracheally to reproduce the model of ALI), NaHS (H₂S donor) +LPS, propargylglycine ［inhibitor of cysathionine-γ-lyase (CSE), PPG］+LPS, CCK-8+LPS, PPG+CCK-8+LPS and CCK-8 group. Animals were sacrificed at 4 h and 8 h after agent instillation. The wet and dry ratio (W/D) of the lung weight was measured and calculated. Morphological changes of lung tissues were observed. H₂S concentration in plasma, malondialdehyde (MDA) content, myeloperoxidase (MPO) and CSE activities in the lung were determined. Furthermore, the level of P-selectin of lung tissue was measured by radioimmunoassay, the CSE mRNA expression in the lung was detected by RT-PCR, and the protein content in bronchoalveolar lavage fluid (BALF) was detected. RESULTS: Compared with control, severe injury of lung tissues and increase in W/D, protein content in BALF, MDA content, MPO activity and P-selectin level in the lung were observed in rats treated with LPS. LPS also lead to a drop in plasma H₂S concentration, lung CSE activity and CSE mRNA expression. Administration of NaHS before LPS could attenuate the changes induced by LPS, while H₂S concentration, CSE activity and CSE mRNA expression were higher than those in LPS group. However, pre-treatment with PPG exacerbated the lung injury induced by LPS, H₂S concentration, CSE activity and CSE mRNA expression were lower than those in LPS and CCK-8 +LPS group, respectively. CONCLUSION: CCK-8 attenuates LPS-induced acute lung injury by means of anti-oxidation and inhibition of PMN adhesion and aggregation, both of which are mediated by endogenous H₂S.     

Porcine surfactant in treatment of LPS-induced early-stage acute lung injury in rats

AIM: To evaluate the therapeutic efficacy of intratracheal instillation of porcine pulmonary surfactant (PPS) in rats with lipopolysaccharides (LPS)-induced early-stage ALI in this study.METHODS: SD rats weighing 200 g-300 g were randomly divided into 4 groups: LPS (1.5 mg·kg-1)+saline,LPS+PPS 100 mg·kg^-1,LPS+PPS 150 mg·kg^-1,LPS+PPS 200 mg·kg^-1.The PaO₂ and PaCO₂,as well as survival rate of rats were examined for 6 h after the start of PPS-instillation.Then,rats were killed and lungs were immediately removed for lung index (LI) and histological analysis.The bronchoalveolar lavage fluid (BALF) was collected for measurement of total protein (TP) contents,TNF-α level and white blood cell(WBC) numbers.RESULTS: Significantly increased PaO₂,reduced mortality rate,decreased total protein and TNF-α contents in BAL,as well as lung index and meliorated histological appearance were observed in three PPS-treated groups compared with group given saline after LPS (P<0.05).The therapeutic effect in PPS150 and PPS200 groups was better than that in PPS100 group.CONCLUSION: Intratracheal PPS instillation provides protective effect on acute lung injury in rats induced by LPS.     

Proportion of CD14+CD16+ monocytes in peripheral blood from patients with type 2 diabetic mellitus and effect of LPS and IL-15 on expression of STAT5 in monocytes

AIM: To characterize the proportion of CD14⁺CD16⁺ monocytes in peripheral blood from type 2 diabetes (T2DM) patients and to observe the response of CD14⁺CD16⁺ monocytes to lipopolysaccharide (LPS) and interleukin-15 (IL-15) for further exploring the potential mechanism of inflammatory immune response in the pathogenesis of T2DM. METHODS: Twenty-eight patients with T2DM and 20 healthy volunteers were enrolled in the study. The peripheral blood was collected for determining the percentage of CD14⁺CD16⁺ monocytes by flow cytometry. The peripheral blood mononuclear cells (PBMC) were isolated and subject to stimulation with LPS and IL-15 for 4 h. The protein expression of STAT5 was detected by Western blotting and the phosphorylated (p)-STAT5 was determined by Western blotting and immunofluorescence. Serum levels of 25-hydroxyvitamin D₃ and IL-6, and the concentrations of IL-6 and monocyte chemoattractant protein-1(MCP-1) in the culture supernatants were assessed by ELISA. Serum level of C-reactive protein (CRP) was measured by immunoturbidimetry. RESULTS: There were positive correlations between the quantity of CD14⁺CD16⁺ monocytes and serum levels of CRP and IL-6 (r=0.394, P<0.05 and r=0.741, P<0.01), while serum 25 (OH) D₃ was negatively correlated with the quantity of CD14⁺CD16⁺ monocytes (r=-0.409, P<0.01), serum CRP(r=-0.479,P<0.01) and serum IL-6 (r=-0.774,P <0.01). After stimulated with LPS and IL-15, PBMC showed significant up-regulation of p-STAT5 protein expression, and significant increases in the supernatant levels of IL-6 and MCP-1 were observed (P<0.05). The expression of p-STAT5 existed in the nucleus.CONCLUSION: These findings suggest that the functional disturbance in monocytes occurs in T2DM, which may be related to insufficiency of vitamin D₃. The aberrant activation of STAT5 signaling pathway underlies the functional abnormalities of the monocytes in T2DM.