Necrostatin-1 prevents skeletal muscle ischemia reperfusion injury by regulating Bok-mediated apoptosis.

BACKGROUND: Receptor interacting serine/threonine kinase 1 (RIPK1) mediates apoptosis by regulating the classic proapoptotic effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak). Although Bcl-2-related ovarian killer (Bok) is structurally similar to Bak and Bax, it is unclear whether it mediates apoptosis in skeletal muscle ischemia reperfusion (IR) injury. We hypothesized that by regulating Bok-mediated apoptosis, inhibiting RIPK1 with necrostatin-1 would reduce skeletal muscle IR injury. METHODS: Rats were randomized into four groups: sham (SM), IR, IR treated with necrostatin-1 (NI), or vehicle dimethyl sulfoxide (DI). For the IR group, the right femoral artery was clamped for 4 hours and then reperfused for 4 hours, and for the NI and DI groups, necrostatin-1 (1.65 mg/kg) and the equal volume of dimethyl sulfoxide were intraperitoneally administered prior to IR induction. The structural damage of muscle tissue and protein expression of Bok, Bcl-2, and cleaved caspase-3 were investigated, and apoptotic cells were identified with terminal dUTP nick-end labeling (TUNEL) staining. In vitro, human skeletal muscle cells (HSMCs) were exposed to 6 hours of oxygen-glucose deprivation followed by normoxia for 6 hours to establish an oxygen-glucose deprivation/reoxygenation (OGD/R) model. To determine the role of Bok, cell viability, lactate dehydrogenase (LDH) release, and flow cytometry were examined to demonstrate the effects of necrostatin-1 and Bok knockdown on the OGD/R insult of HSMCs. RESULTS: Necrostatin-1 pretreatment markedly reduced IR-induced muscle damage and RIPK1, Bok, and cleaved caspase-3 expression, whereas upregualted Bcl-2 expression (p < 0.05). Furthermore, necrostatin-1 prevented mitochondrial damage and decreased TUNEL-positive muscle cells (p < 0.05). In vitro, HSMCs treated with necrostatin-1 showed reduced Bok expression, increased cell viability, and reduced LDH release in response to OGD/R (p < 0.05), and Bok knockdown significantly blunted the OGD/R insult in HSMCs. CONCLUSION: Necrostatin-1 prevents skeletal muscle from IR injury by regulating Bok-mediated apoptosis.

[The regulatory role of autophagy in rats lung ischemia/reperfusion injury].

Objective: To investigate the role of cell autophagy in lung ischemia/reperfusion injury in rats. Methods: Forty SD rats were randomly divided into 5 groups (n=8): ①Sham operated group (sham group):just open rat chest for 3.5 h; ②Ischemia/reperfusion group (I/R group):after open chest, clamp pulmonary hilus for 0.5h then reperfusion for 3 h; ③Solvent group (DMSO group): intraperitoneal injection of DMSO solution for 1h before operation; ④Autophagic inhibitor group (3-MA group); ⑤Autophagic agonist group (Rap group): intraperitoneal injection of autophagic agonist rapamycin before operation; the rest operations of DMSO, 3-MA and Rap groups are the same as that of I/R group. At the end of the experiment, the rats were killed by euthanasia-killing. The lung tissues were collected and the wet/dry weight ratio (W/D) and total lung water content (TLW) of the lung tissues were detected. The lung tissue structure and cell ultramicro morphology were observed by light microscopy and electron microscopy and the injuried alveolar rate(IAR) was calculated. The autophagy-related protein expressions were detected by Western blot. Results: Compared with sham group, the levels of W/D, TLW and IAR were increased, the expressions of autophagy related protein and p-AMPK, Beclin 1, LC3 II were also increased in other four groups, while the protein expressions of p-mTOR and p62 were decreased significantly (P＜ 0.05 or P＜0.01). Under the light microscope, the other groups of lung tissue had edema and exudation in varying degrees, the structure of alveoli was disordered, the ultrastructural damage of cells was aggravated under the electron microscope, and autophagosome could be observed. Compared with DMSO group, the expressions of autophagy related protein, the levels of W/D, TLW and IAR in 3-MA group were decreased (P＜0.05 or P＜0.01), the edema of lung interstitial was lighter, and less cells were found in alveolar cavity. Ultrastructural damage was also lighter and with less autophagosome. Besides, there was no significant difference among I/R, DMSO and Rap groups (P＞0.05). Conclusion: Autophagy can be activated during ischemia/reperfusion in rats to induce lung injury.

[Effect of dexmedetomidine on apoptosis and CHOP in hypoxia/reoxygenation injury A549 cell].

OBJECTIVES: To investigate the effects of dexmedetomidine (Dex) on injury of A549 cells induced by hypoxia/reoxygenation(H/R)and the influence of C/EBP homologous protein (CHOP) expression. METHODS: Logarithmic growth phase A549 cells(it originated from alveolar type Ⅱ epithelial cell line) were randomly divided into 4 groups (n=10):normoxic control group (N), Dex group (D), hypoxia/reoxygenation group (H), hypoxia/reoxygenation + Dex group(HD). At the beginning of modeling, 1 nmol/L Dex was puted into D and HD groups. N and D groups were cultured in the normoxic incubator for 30 h. H and HD group were incubated in the anoxic cultivation for 6 h, fo llowed by normoxic culture for 24 h. Then A549 cells were observed under the inverted microscope to observe the morphological changes. Cell activity was detected by cell counting Kit-8(CCK-8) and the apoptosis index(AI) was detected by in situ end labeling (TUNEL) method. The expression of CHOP、glucose-regulated protein of molecular weight 78 kDa (Grp78)、cysteinyl aspirate-specificprotease-3 (caspase-3) protein and CHOP、Grp78 mRNA were detected by Western blot and RT-PCR. RESULTS: Compared with N group, the number of adherent cells in H group decreased significantly, and cell morphology changed. The absorbance value in H group decreased obviously (P<0. 01). The AI value and expression of CHOP, Grp78, caspase-3 proteins and CHOP, Grp78 mRNA were significantly increased (P<0.01). Compared with H group, the cell damage in HD group was decreased, the absorbance value increased (P<0.01), the number of apoptosis cells decreased relatively (P<0.01), the expression of CHOP, caspase-3 protein and CHOP mRNA decreased (P<0. 01). CONCLUSIONS: Dex has notable effects against H/R injury, which may be related to effective inhibition of apoptosis mediated by the CHOP's signal path.

[The role of endoplasmic reticulum stress in pulmonary hypertension in rat induced by chronic hypoxia and hypercapnia].

OBJECTIVE: To observe the pulmonary vascular remodeling in rats with pulmonary hypertension induced by hypoxia and hypercapnia, and to explore the role of endoplasmic reticulum stress in pulmonary hypertension. METHODS: Forty SD rats were random-ly divided into four groups:normoxic control group (N), hypoxia hypercapnia group (HH), ERS inhibitor 4-phenylbutyric acid group (4-PBA), endoplasmic reticulum stress (ERS) pathway agonist tunicamycin group (TM), ten rats in each group.The mean pulmona-ry artery pressure (mPAP), mean carotid artery pressure (mCAP) and right ventricular hypertrophy index of rats in each group were measured.Pulmonary artery smooth muscle cells were identified by immunofluorescence α-smooth muscle actin (α-SMA).Morphologi-cal changes of lung tissue and pulmonary artery were observed by electron microscope.The apoptotic index of pulmonary artery smooth muscle cells in each group was detected by TUNEL.Reverse transcription polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), c-Jun N-terminal kinase (JNK) and cysteinyl aspartate specific proteinase-12 (caspase-12) mRNA and protein in each group. RESULTS: ①Compared with the N group, the mPAP, the ratio of right ventricle weight to left ventricle plus ventricular septum weight[RV/(LV+S)]and the ratio of pulmonary artery wall area to total tube area (WA/TA) were increased (P<0.01), and the ratio of pulmonary artery luminal area to total tube area (LA/TA) were decreased (P<0.01), pulmonary artery smooth muscle cell apoptosis index were decreased (P<0.05 or P<0.01) in HH group, 4-PBA group and TM group.ERS related protein and mRNA expressions were increased, the differences were statistically significant.②Compared with the HH group, the mPAP, [RV/(LV+S)]and WA/TA of 4-PBA group were decreased (P <0.01), LA/TA and pulmonary artery smooth muscle cell apoptosis index were increased (P<0.01, P<0.05).The expressions of ERS related protein and mRNA were all decreased (P<0.05 or P<0.01).③Compared with the HH group, the mPAP, [RV/(LV+S)]and WA/TA of TM group were increased (P<0.05 or P<0.01), pulmonary artery middle layer thickened, LA/TA and pulmonary artery smooth muscle cell apoptotic index were decreased (P<0.01).ERS related protein and mRNA expressions were increased with statistical significance except GRP78 protein. CONCLUSIONS: Pulmonary vascular remodeling in rats with pulmonary hypertension induced by hypoxia and hypercapnia may be related to the excessive proliferation of pulmonary artery smooth muscle cells and too little apopto-sis;ERS related factors (JNK, caspase-12 and CHOP) are involved in the regulation of pulmonary hypertension induced by hypoxia hypercapnia.

[Effects of Dexmedetomidine on the levels of proinflammatory mediators IL-1ßand TNF-α in pulmonary ischemia/reperfusion injury rats and the mechanisms].

OBJECTIVE: To evaluate the effects and mechanism of the Dexmedetomidine on the levels of proinflammatory mediators interleukin 1 beta (IL-1ß) and tumor necrosis factor-α(TNF-α) in ischemia/reperfusion(I/R)rats. METHODS: Fifty healthy SPF male SD rats, 250~310 g,8~12 weeks,were randomly divided into five groups(n=10):sham operation group(sham group),I/R group, dexmedetomidine group(Dex group), atipamezole group(Atip group), dexmedetomidine plus atipamezole(Dex+Atip group). The I/R model was established by clipping hilus of left lung for 30 min and then reperfusion for 2 h. Dex group, Atip group and Dex + Atip group were performed by intraperitoneal injection dexmedetomidine(20 µg/kg),atipamezole(250 µg/kg),Dexmedetomidine(20 µg/kg)+atipamezole(250 µg/kg)respectively 30 min in advance before hilus of left lung was clipped, the rest of the process was the same with I/R group. After the experiment the rats were killed and the left lung tissues to determine the lung wet/dry weight(W/D) and total lung water content(TLW); Ultra structure of lung tissues were observed under light microscope and electron microscope; IL-1ß and TNF-α levels were determined by using ELISA. RESULTS: Compared with the sham group, the W/D、TLW、IL-1ß and TNF-α in other groups were increased significantly (P<0.05). The structure damages of lung tissues observed under light microscope and electron microscope in other groups were more serious than that of sham group. Compared with I/R、Atip、Dex+Atip group, the levels of W/D、TLW,IL-1ß and TNF-α in Dex group were lower (P<0.05), the structure damages of lung tissues observed under light microscopy and electron microscope in Dex group were slighter. There was no significant difference of the above parameters among I/R、Atip、Dex+Atip group. CONCLUSIONS: Dexmedetomidine can alleviate ischemia/reperfusion injury in rat lung through lowering the level of proinflammatory mediators IL-1ß and TNF-α,the possible mechanism may be through stimulation of α2 adrenaline receptors.

[Effect of dexmedetomidine on renal injury induced by lung ischemia/reperfusion in mice].

OBJECTIVE: To evaluate the effect of dexmedetomidine(Dex) on renal injury induced by lung ischemia/reperfusion(I/R) in mice. METHODS: Fifty healthy SPF male C57BL/6J mice, weighing 20 g~24 g,aged 8~10 weeks,were randomly divided into five groups(n=10 each):sham operation group(sham group),lung ischemia/reperfusion group(I/R group), lung ischemia/reperfusion and normal saline group (NS group), dexmedetomidine group(Dex group), dexmedetomidine and atipamezole group (DA group). Lung ischemia/reperfusion model was established by occlusion of the left pulmonary artery for 30 min followed by 180 min reperfusion in mice. In Dex and DA groups, dexmedetomidine 20 µg/kg and dexmedetomidine 20 µg/kg plus atipamezole 250 µg/kg were injected intraperitoneally respectively at 30 min before establishment of the model, isopyknic normal saline instead of Dex were injected intraperitoneally in NS group. After the experiment the mice were killed and plasma IL-1 beta and tumor necrosis factor α(TNF-α) concentration were detected by ELISA; the renal tissues were harvested to observe ultra structure under electron microscope. RESULTS: Compared with sham group, the concentrations of IL-1ß and TNF-α in other groups were increased significantly and the structure damages of renal tissues observed under electron microscope in other groups were more serious than those of sham group. Compared with I/R group, NS groups and DA group, the concentrations of IL-1ß and TNF-α in Dex group were significantly lower(P<0.05)and the structure damages of renal tissues observed under electron microscope in Dex group were slighter. CONCLUSIONS: Dexmedetomidine pretreatment can attenuate renal injury induced by lung ischemia/reperfusion and the mechanism may be related to inhibition of inflammatory responses.

[The effects of YHTJF on lung ischemia/reperfusion injury in mice].

OBJECTIVE: To explore whether yiqi huoxue tongluo jiedu fang (YHTJF, Traditional Chinese Medicine) alleviates the injury during lung ischemia/reperfusion (I/R) in mice through inhibiting oxidative stress or not. METHODS: C57BL/6J male mice (n=70) were randomly divided into 7 groups:control (C), carboxyl methyl cellulose-Na(CMC·Na) + normal control (CC), carboxyl methyl cellulose-Na + sham (CS), carboxyl methyl cellulose-Na + I/R (CIR), carboxyl methyl cellulose-Na + YHTJF-Low, CMC-Na + YHTJF-Middle, CMC-Na + YHTJF-High (CYL, CYM, CYH). The mice in CYL, CYM and CYH group were treated with YHTJF by intraperitoneal injection every day, while the carboxyl methyl cellulose-Na was administered with the same volume of CYL in CC, CS and CIR group. After 3 h-reperfusion, the left lung tissues were harvested to determine the lung wet/dry weight (W/D), the total lung water content (TLW), and the index of quantita-tive evaluation for alveolar damage (IQA). Morphological observation and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) were applied to evaluate the structural changes and the apoptosis index (AI) of the lung tissues. The expressions superoxide of dis-mutase(SOD), malondialdehyde(MDA) and myeloperoxidase(MPO) in the lung tissues were detected by kits. RESULTS: Compared with group C, the W/D, TLW, IQA, AI, lung tissue structural changes, and the expressions of MDA and MPO in group I/R were increased obviously (P < 0.01), and the expression of SOD was decreased, while there was no significant difference between group CC and CS. Compared with group I/R, the parameters of these experiments in group CYL, CYM, CYH were all decreased, and the expression of SOD was increased, while the reduction in group CYM was the most remarkable among them (P < 0.01). CONCLUSIONS: YHTJF may attenuate the I/R injury of the lung by the inhibition of apoptosis via ROS pathway.

[Effect of dexmedetomidine on expression of endoplasmic reticulum stress-related Caspase-12 in lung ischemia/reperfusion injury mice].

OBJECTIVE: To investigate the effect of dexmedetomidine (DEX) on expression of endoplasmic reticulum stress (ERS)-related cysteinyl aspirate specific proteinase-12 (Caspase-12) in lung ischemia/reperfusion (I/R) injury mice. METHODS: Forty C57BL/6J mice were randomly divided into 4 groups:sham operation group (sham group),ischemia/reperfusion injury group (I/R group), normal salinecontrol group (NS group), ischemia/reperfusion + dexmedetomidine group (DEX group). Dexmedetomidine was infused intraperitoneally into the mice to stablish situ left pulmonary I/R injury mouse model. In NS group, the isometric dexmedetomidine was replaced by normal saline,other operations were as the same as the DEX group. After reperfusion 3 hours, the lung tissue wet/dry weight (W/D), the total lung water content (TLW) of the left lung tissues were determined. The lung tissue morphology changes were observed by light microscopy and the damage assessment(IQA) was taken. The structure changes and the apoptosis index (AI) of the lung tissues were evaluated by TUNEL method. The protein and mRNA expression of Caspase-12 and grp78 in lung tissues were detected by Western blot and reverse translate-PCR. RESULTS: Compared with the sham group, the W/D, TLW, IQA, AI, lung tissue structure damages, and the expression of Caspase-12 and grp78 protein and mRNA obviously raised both in I/R group and NS group (P<0.01 or P<0.05). Compared with I/R group, the W/D, TLW, IQA, AI of DEX group were all decreased, the demaged lung tissue morphology changes were significantly reduced, the protein and mRNA expression level of Caspase-12 and grp78 in DEX group were decreased (P<0.01). CONCLUSIONS: DEX can effectively relieve the lung I/R injuries in mice, which maybe associated with inhibition of pneumocyte apoptosis induced by ERS-related Caspase-12 pathway.

[Ischemic postconditioning attenuates pneumocyte apoptosis after lung ischemia/reperfusion injury via inactivation of p38 MAPK].

OBJECTIVE: To investigate the role of p38 MAPK on ischemic postconditioning (IPO) attenuating pneumocyte apoptosis after lung ischemia/reperfusion injury (LIRI). METHODS: Forty adult male SD rats were randomly divided into 5 groups based upon the intervention (n = 8): control group (C), LIR group (I/R), LIR + IPO group (IPO), IPO + solution control group (D), IPO + SB203580 group (SB). Left lung tissue was isolated after the 2 hours of reperfusion, the ratio of wet lung weight to dry lung weight (W/D), and total lung water content (TLW) were measured. The histological structure of the left lung was observed under light and electron transmission microscopes, and scored by alveolar damage index of quantitative assessment (IQA). Apoptosis index (AI) of lung tissue was determined by terminal deoxynuleotidyl transferase mediated dUTP nick end and labeling (TUNEL) method. The mRNA expression and protein levels of and Bax were measured by RT-PCR and quantitative immunohistochemistry (IHC). RESULTS: Compared with C group, W/D, TLW, IQA, AI and the expression of Bax of I/R were significantly increased, the expression of Bcl-2 and Bcl-2/Bax were significantly decreased (P < 0.05, P < 0.01), and was obviously morphological abnormality in lung tissue. Compared with I/R group, all the indexes of IPO except for the expression of Bcl-2 and Bcl-2/ Bax were obviously reduced, the expression of Bcl-2 and Bcl-2/Bax were increased (P < 0.05, P < 0.01). All the indexes between D and IPO were little or not significant( P > 0.05). The expression of Bcl-2 and Bcl-2/Bax of SB were significantly increased and other indexes were reduced than those of IPO (P < 0.05, P < 0.01). CONCLUSION: IPO may attenuate pneumocyte apoptosis in LIRI by inactivation of p38 MAPK, up-regulating expression of Bcl-2/Bax ratio.

[Effect of siRNA silencing the role of JNK gene in excessive endoplasmic reticulum stress on lung ischemia/reperfusion injury].

OBJECTIVE: To investigate the effect of siRNA silencing the role of C-Jun N-terminal Kinase (JNK) gene in excessive endoplasmic reticulum stress on lung ischemia/reperfusion injury. METHODS: Mouse model of pulmonary ischemia reperfusion injury (PIRI) in situ was established with unilateral lung in vivo. Seventy experimental mice were randomly allocated into seven groups (n = 10): Sham group (Sham group), ischemia reperfusion group (I/R), PBS+ Lipofectamine2000TM transfection reagent group (I/R + PBS+ Lipo group), negative control group (I/R+ SCR group), JNK-siRNA group (I/R + siRNA(JNK1), siRNA(JNK2), siRNA(JNK3)). Mice were euthanized after experimental time out, and left lung tissue was extracted. Wet/dry lung weight ratio (W/D) and total lung water content (TLW) were tested. Light microscope, alveolar damage quantitative evaluation index (IQA) and electron microscope were observed. The expression levels of JNK and glucose regulatex protein(GRP78) were detected by RT-PCR and Western blot. Apoptosis of lung tissue was determined by TUNEL. RESULTS: Compared with Sham group, all indicators above of I/R + PBS + Lipo group and I/R + SCR group were significantly increased (P < 0.01), and compared with I/R group, those indicators of the three groups all had no notable difference; those indicators were not statistically different between I/R + PBS + Lipo group and I/R + SCR group, and compared to the three groups, the above indicators in JNK-siRNA group were lower (P < 0.05, P < 0.01) except that the expression levels of GRP78 was not statistically different. CONCLUSION: I/R induces excessive ERS in lung tissue, in which JNK pathway participates in apoptosis, leading to lung tissue injury.

[Effect of Xuebijing injection on TLR4--NF-kappaB--IL-1beta pathway of myocardial hypoxia/reoxygenation in rats].

OBJECTIVE: To investigate the role of Xuebijing injection(XBJI, traditional Chinese medicine), in inhibiting TLR4--NF-kappaB--IL-1beta pathway of myocardial hypoxia/reoxygenation in rats. METHODS: Thirty six male SD rats (280 +/- 30) g were randomly divided into six groups (n = 6): normal group (N group), balanced perfusion group (BP group), model group (M group), low dose XBJI group (XBJI(L) group), middle dose XBJI group (XBJI(M) group), high dose XBJI group (XBJI(H) group). By Langendorff isolated heart perfusion device to establish the model of myocardial hypoxia/reoxygenation in rats. ELISA was used to detect the concentration of interleukin-1beta (IL-1beta); Western blot was used to detect the expression of nuclear factor kappa B p65 (NF-kappaB p65) protein and toll like receptor 4 (TLR4) protein; and RT-PCR to determine the expression of NF-kappaB p65 mRNA and TLR4 mRNA;To observe microstructure changes of hypoxia/reoxygenation myocardial by light microscopy. RESULTS: Compared with M group, the IL-1beta concentration, NF-kappaB p65 and TLR4 protein,NF-kappaB p65 and TLR4 mRNA of XBJIL group, XBJI(M) group, XBJI(H) group expression decreased in varying degrees,and decreased most obviously all in XBJI(M) group (P < 0.05, P < 0.01); Myocardical structural damage was serious in M group, and improved after treatment XBJI, the most obvious was the XBJI(M). CONCLUSION: Different dose of XBJI can inhibit TLR4--NF-kappaB--IL-1beta signal transduction pathway and reduce several inflammatory reaction after myocardial hypoxia/reoxygenation injury, the 4 ml/100 ml of XBJI is the best.

[Effects of ischemic postconditioning on pneumocyte apoptosis after lung ischemia/reperfusion injury in rats].

OBJECTIVE: To investigate the effects of ischemic postconditioning (IPostC) on pneumocyte apoptosis after lung ischemia/reperfusion injury in rats. METHODS: Adult male SD rats were randomly divided into 3 groups based upon the intervention (n = 8): control group (C), lung ischemic reperfusion group (LIR), LIR+ IPostC group (IPostC). At the end of the experiment, blood specimens drawn from the arteria carotis were tested for the content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and myeloperoxidase (MPO); the pneumocyte apoptosis index (AI) was achieved by tennrminal deoxynucleotidyl transferase mediated dUTP nick end abeling (TUNEL); the expression of Bcl-2, Bax protein in lung tissue was accessed by quantitative immunohistochemistry (MHC) and Bcl-2, Bax mRNA by RT-PCR. RESULTS: IPostC could significantly attenuate the MDA level, MPO activity and improve SOD activity in blood serum which was comparable to I/R and significantly reduced the number of TUNEL-positive cells compared with I/R group, expressed as Al (% total nuclei) from (39.0 +/- 3.46) to (8.0 +/- 0.88) (P < 0.01). The protein and mRNA expression of Bcl-2 and Bax showed that IPO significantly attenuated the ischemia/reperfusion-upregulated expression of Bax protein but improved the expression of Bcl-2 that improved the Bcl-2/Bax ratio (P < 0.01) . CONCLUSION: IPostC may attenuate pneumocyte apoptosis in LIRI by up-regulating expression of Bcl-2/Bax ratio and by inhibiting oxidant generation and neutrophils filtration.

[Calcium-activated chloride channels are involved in two-phase hypoxic pulmonary vasoconstriction in rat pulmonary arteries].

The aim of the present study was to investigate the roles of calcium-activated chloride channels (Cl(Ca)) in the two-phase hypoxic pulmonary vasoconstriction (HPV). The second pulmonary artery branches were dissected from male Sprague-Dawley rats, and the changes in vascular tone were measured by using routine blood vascular perfusion in vitro. The result showed that, under normoxic conditions, Cl(Ca) inhibitors (NFA and IAA-94) significantly relaxed second pulmonary artery contracted by norepinephrine (P < 0.01), but merely had effects on KCl-induced second pulmonary artery contractions. A biphasic contraction response was induced in second pulmonary artery ring pre-contracted with norepinephrine exposed to hypoxic conditions for at least one hour, but no biphasic contraction was observed in pulmonary rings pre-contracted with KCl. NFA and IAA-94 significantly attenuated phase II sustained hypoxic contraction (P < 0.01), and also attenuated phase I vasodilation, but had little effect on phase I contraction. These results suggest that Cl(Ca) is an important component forming phase II contraction in secondary pulmonary artery, but not involved in phase I contraction.

[Effect of Xuebijing Injection on TLR4-NF-κB-TNF-α pathway of rats' myocardial anoxia/reoxygenation].

OBJECTIVE: To explore the role of Xuebijing Injection (XBJI) in inhibiting inflammatory factors associated with anoxia/reoxygenation myocardial inflammatory response of rats. METHODS: Totally 36 healthy male Sprague-Dawley rats, 280 ± 30 g were randomly divided into six groups, i.e., the normal control group (N group), the balanced perfusion group (BP group),the model group (M group),the low dose XBJI group (XBJI(L) group), the middle dose XBJI group (XBJI(M) group),and the high dose XBJI group (XBJI(H) group), 6 in each group. The myocardial anoxia/reoxygenation rat model was established by Langendorff isolated heart perfusion. The concentration of TNF-α in the myocardial tissue was detected by ELISA. The expression of nuclear factor kappa B p65 (NF-κB p65) protein and Toll like receptor 4 (TLR4) protein were detected using Western blot. The expression of NF-κB p65 mRNA and TLR4 mRNA was detected by RT-PCR. Ultrastructural changes of anoxia-reoxygenation rats' heart muscle were observed under transmission electron microscope. RESULTS: Compared with the M group,the TNF-α concentration, expression levels of NF-κB p65 protein and mRNA, TLR4 protein and mRNA decreased to various degrees in the XBJI(L) group, the XBJI(M) group, and the XBJI(H) group. The TNF-α expression level decreased most significantly in the XBJI(L), group (P < 0.01), while other indices decreased most obviously in the XBJI(M) group (P < 0.01, P < 0.05). Expression levels of NF-κB p65 and TLR4 protein were obviously lower in the XBJI(M) group than in the XBJI(L) group (P < 0.05). There was no statistical difference in other indices among the three XBJI groups (P > 0.05). Myocardial fibers were loose and broken with disappearance of transverse striation, and mitochondrial cristae was dissolved and severely damaged in the M group. The aforesaid condition was improved after treated by XBJI, with the most obvious effect obtained in the XBJI(M) group. CONCLUSIONS: Different doses of XBJI could attenuate inflammatory reactions after myocardial anoxia/reoxygenation rats' heart muscle through inhibiting TLR4-NF-κB-TNF-α signal transduction pathway. The best effect could be obtained by 4 mL/100 mL XBJI.

[Expression of steroidogenic enzymes in the rat model of polycystic ovary syndrome].

The aim of the present study was to investigate the expression changes of three steroidogenic enzymes in the polycystic ovary syndrome (PCOS). Thirty Sprague-Dawley (SD) rats were randomly divided into normal control (NC) group and PCOS group. PCOS rat model was established by DHEA injection. The serum levels of progesterone, estrogen and testosterone were measured by immunoradioassay or enzyme immunoassay. The cellular distributions of 3ß-hydroxy steroid dehydrogenase (3ß-HSD), 17ß-hydroxy steroid dehydrogenase (17ß-HSD) and cytochrome P450 aromatase (P450arom) in ovaries were detected by immunohistochemistry. The expression levels of 3ß-HSD, 17ß-HSD and P450arom were detected by RT-PCR and Western blot. The results showed that the serum levels of estrogen and testosterone of PCOS group were significantly higher than those of the NC group. There was no significant difference of serum progesterone level between the PCOS and NC groups. Compared with the NC group, the PCOS group showed increased mRNA and protein expressions of both 3ß-HSD and 17ß-HSD, as well as reduced P450arom mRNA and protein expressions. These results suggest that 3ß-HSD and 17ß-HSD, but not P450arom, may participate in the ovarian hormonal regulation in the present rat model of PCOS.

[Protective effects and mechanism of SP600125 on lung ischemia/reperfusion injury in rats].

OBJECTIVE: To investigate the protective effects and mechanism of SP600125-specificity inhibitor of c-Jun N-terminal kinase (JNK)on lung ischemia /reperfusion injury in rats. METHODS: The unilateral lung ischemia/reperfusion model was replicated in vivo. Rats were randomly divided into three groups (n = 10): control group, ischemia/reperfusion group ( I/R group) and ischemia/reperfusion + SP600125 group (SP600125 group). The lung tissues sampled at the end of each experiment were assayed for wet/dry weight ratio (W/D),the injured alveoli rate (IAR), the expression of phosphorylation JNK (p-JNK) and JNK protein were detected by Western blot, the expression of Bcl-2, Bax, Caspase3 protein were detected by immunocytochemistry techniques, the pneumocyte apoptosis index (AI) was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end abeling(TUNEL), the ultrastructure changes were observed under electron microscope. RESULTS: Compared to I/R group, the expression of p-JNK, Bcl-2, Bax and caspase-3 protein were markedly decreased (all P < 0.01), the expression of Bcl-2 protein and the ratio of Bcl-2/Bax were markedly increased in SP600125 group(all P < 0.01). The value of AI, W/D, IAR showed significantly lower than those in I/R group (all P <0.01). Meanwhile, light morphological and ultrastructure injury were found in SP600125 group. CONCLUSION: SP600125 can suppress JNK signal pathway, up-regulate the ratio of Bcl-2/Bax to inhibit Caspase-3 dependent apoptosis, so that it protects lung tissue from ischemia/reperfusion injury.

Diabetes increases inflammation and lung injury associated with protective ventilation strategy in mice.

BACKGROUND: Mechanical ventilation may paradoxically cause lung injury. Protective mechanical ventilation strategy utilizing low tidal volume and high frequency has been shown to attenuate inflammation and reduce mortality in non-diabetic patients. The purpose of this present study was to observe the effects of diabetes on inflammation and lung injury in mice with protective ventilation strategy. METHODS: Forty mice were included in our study. The mice in Group Dia-MV and Con-MV were subjected to 4 hour-ventilation. And the mice in Group Dia-SB and Con-SB were exposed to room air breathing spontaneously for 4h. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), superoxide dismutase (SOD) and malondialdehyde (MDA) levels in serum were detected and the expression of inflammatory cytokine mRNA was also determined in lung tissue. Lung damage was assessed using a modified lung injury score. RESULTS: The serum levels of TNF-α, IL-6, and IL-10 in Group Dia-MV were significantly higher than those in Group Dia-SB or Group Con-MV or Group Con-SB (P<0.05). Quantitative RT-PCR analysis of pro-inflammatory cytokines in lung homogenates presented similar results. The mice in Group Dia-MV suffered obvious lung histological changes, whose lung injury scores were significantly higher in Group Dia-SB as compared to Group Con-SB , Group Con-MV or Group Dia-SB (P<0.05). CONCLUSIONS: Diabetes increased the inflammation reaction and associated lung injury in mice in spite of the protective mechanical ventilation strategy based on low tidal volumes and high frequency.

[The relationship between endogenous hydrogen sulfide system and pulmonary hypertension induced by hypoxic hypercapnia].

OBJECTIVE: To investigate the changes of the endogenous hydrogen sulfide(H2S) system in pulmonary hypertension induced by hypoxic hypercapnia (HHPH) in rats and approach the possible mechanisms. METHODS: 20 SD rats were randomly divided into control group (C) and hypoxic hypercapnia group (HH) (n=10). The changes of hemodynamics and the right ventricle/left ventricle + septum (RV/LV + SP) were measured. The ratio of vessel wall area and total area (WA/TA) of arteriae pulmonalis were observed under lightmicroscope. By using TdT-mediated dUTP nick end labeling (TUNEL) and immunocytochemistry techniques, apoptosis index (AI) and expression of Bcl-2, Bax protein in arteriae pulmonalis were tested. Plasma level of H2S and activity of H2S generating enzymes in homogenates of rat lung tissue were evaluated by sensitive modified sulfide electrode method. Cystathionine-gamma-lyase (CSE) mRNA in lung tissues was determined by RT-PCR. RESULTS: The level of mean pulmonary arterial pressure(mPAP), WA/TA and RV/LV + SP were significantly higher in HH group than those in C group (P < 0.05 or P < 0.01). Compared with those in C group, the AI of arteriae pulmonalis in HH group were significantly lower; the expression of Bcl-2 protein increased while that of Bax protein decreased, and the ratio of Bax/Bcl-2 went up obviously (all P < 0.01). Plasma level of H2S, the activity of H2S generating enzymes and CSE mRNA in HH group were significantly lower than those in C group (all P < 0.01). Plasma level of H2S, the activity of H2S generating enzymes, CSE mRNA each was closely positively related to Al while inversely related to mPAP and Bcl-2/Bax (all P < 0.01). CONCLUSION: The endogenous hydrogen sulfide system is closely related to pulmonary hypertension induced by hypoxic hypercapnia. The depression of the H2S/CSE system in HHPH may help increase the ratio of Bcl-2/Bax, inhibit apoptosis of pulmonary artery smooth muscle cells and finally result in the formation of pulmonary hypertension.

[Effects of ambroxol combined with low-dose heparin on TNF-alpha and IL-1beta in rabbits with acute lung injury].

OBJECTIVE: To investigate the intervention and mechanism of ambroxol combined with low-dose heparin on oxidative stress, TNF-alpha and IL-1beta in rabbits with acute lung injury (ALI). METHODS: Twenty-four healthy Japanese rabbits were randomly divided into three groups: (1) Normal saline control group (NC), (2) Oleic acid injury group (OA), (3) Ambroxol + low-dose heparin therapy group (AH). After the success of ALI model, AH group was injected ambroxol + low-dose heparin, while the NC group and OA group were injected the same dose of normal saline by the same method. Arterial oxygen tension (PaO2), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) at different time points were determined. The pathological manifestation of both side lungs was observed at the end of expeiment. The activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), xanthine oxidase (XO) and the content of malondialdehyde (MDA) in bronchoalveolar lavage fluid (BALF) and lung tissue homogenate were tested. The apoptosis index was detected. The lung wet/dry (W/D) ratio was calculated. The pathological changes in lung tissue were observed by light microscopy, and the ultrastructural changes of lung tissue were observed by electron microscopy. RESULTS: (1) The instructive injury induced by ALI observed under electron microscope and light microscope and W/D was decreased significantly in AH group. (2) PaO2 was improved significantly in AH group, compared with that in OA group (P < 0.01). (3) The activity of GSH-Px and SOD in AH group increased significantly (P < 0.01 or P < 0.05) but the activity of XO and the content of MDA decreased significantly (P < 0.01), compared with those in OA group. (4) Except the content of IL-1beta in serum before treatment, the content of IL-1beta and TNF-alpha in serum, BALF, lung tissue homogenate of OA group increased significantly (P < 0.01), and those were obviously improved in AH group. (5) Apoptosis index (AI) in AH group decreased significantly (P < 0.01) compared with that in OA group. CONCLUSION: In ALI induced by OA, IL-1beta and TNF-alpha increases significantly and involved in the occurrence and development of ALI. Ambroxol combined with low-dose heparin can reduce lung cells oxidative stress to inhibit the release of IL-1beta and TNF-alpha, which play a role in the treatment of ALI.

Ischemic preconditioning attenuates pulmonary dysfunction after unilateral thigh tourniquet-induced ischemia-reperfusion.

BACKGROUND: Acute lung injury is a recognized complication of lower limb ischemia-reperfusion that has been demonstrated experimentally and in the clinical setting of aortic surgery. The application of a tourniquet can cause lower limb ischemia-reperfusion in orthopedic surgery. We studied the effect of unilateral thigh tourniquet-induced lower limb ischemia-reperfusion on pulmonary function, and the role of ischemic preconditioning in attenuating pulmonary dysfunction. METHODS: Thirty ASA I or II patients scheduled for lower extremity surgery were randomized into 2 groups: a limb ischemia-reperfusion group with tourniquet application (ischemia-reperfusion group, n = 15) and an ischemia preconditioning group (preconditioning group, n = 15), in which patients received 3 cycles of 5 minutes of ischemia, alternating with 5 minutes of reperfusion before extended use of the tourniquet. Blood gas, plasma malondialdehyde, and serum interleukin-6 (IL-6), IL-8, and IL-10 levels were measured just before tourniquet inflation, 1 hour after inflation and 2 hours, 6 hours, and 24 hours after tourniquet deflation. Arterial-alveolar oxygen tension ratio, alveolar-arterial oxygen tension difference, and respiratory index also were calculated. RESULTS: In comparison with the baseline values, arterial Po(2) and arterial-alveolar oxygen tension ratio were decreased, while alveolar-arterial oxygen tension difference and respiratory index were increased significantly 6 hours after tourniquet deflation in both groups (P < 0.01). However, these changes were less significant in the ischemic preconditioning group than those in the lower limb ischemia-reperfusion group (P < 0.01). Similarly, the increases in the malondialdehyde, IL-6, and IL-8 from 2 hours to 24 hours after release of the tourniquet in the lower limb ischemia-reperfusion group were attenuated by ischemic preconditioning. CONCLUSIONS: Pulmonary gas exchange is impaired after lower limb ischemia-reperfusion associated with the clinical use of a tourniquet for lower limb surgery. Ischemic preconditioning preceding tourniquet-induced ischemia attenuates lipid peroxidation and systemic inflammatory response and mitigates pulmonary dysfunction.