Panax notoginseng saponins improve monocrotaline-induced pulmonary arterial hypertension in rats by inhibiting ADAM10/Notch3 signaling pathway / 生理学报

In this study, we investigated the effects of Panax notoginseng saponins (PNS) on pulmonary vascular remodeling and ADAM10/Notch3 pathway in pulmonary arterial hypertension (PAH). PAH rat model was established, and male Sprague Dawley (SD) rats were randomly divided into control group, monocrotaline (MCT) group and MCT+PNS group, with 10 rats in each group. Rats in the control group were intraperitoneally injected with equal volume of normal saline. Rats in the MCT group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with the same volume of normal saline every day. Rats in the MCT+PNS group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with 50 mg/kg PNS every day. The modeling time of each group lasted for 21 days. After the model was established, the mean pulmonary artery pressure (mPAP) was measured by right heart catheterization technique, the right ventricular hypertrophy index (RVHI) was calculated, the microscopic morphology and changes of pulmonary vascular wall were observed by HE and Masson staining, and the expressions of ADAM10, Notch3, Hes-1, P27, PCNA, Caspase-3 proteins and mRNA in pulmonary vascular tissue of rats were detected by Western blot and qPCR. The expression and localization of Notch3 and α-SMA were detected by immunofluorescence staining. The protein expression of ADAM10 was detected by immunohistochemical staining. The results showed that compared with the control group, mPAP, RVHI, pulmonary vessels and collagen fibers in the MCT group were significantly increased, the expressions of ADAM10, Notch3, Hes-1, and PCNA protein and mRNA were significantly increased, while the expressions of P27 and Caspase-3 protein and mRNA were decreased significantly. Compared with the MCT group, mPAP and RVHI were significantly decreased, pulmonary vessels were significantly improved and collagen fibers were significantly reduced, the expressions of protein and mRNA of ADAM10, Notch3, Hes-1, and PCNA were decreased in MCT+PNS group, but the expressions of protein and mRNA of P27 and Caspase-3 were increased slightly. The results of immunofluorescence showed that Notch3 and α-SMA staining could overlap, which proved that Notch3 was expressed in smooth muscle cells. The expression of Notch3 in the MCT group was increased significantly compared with that in the control group, while PNS intervention decreased the expression of Notch3. Immunohistochemical staining showed that compared with the control group, the amount of ADAM10 in the MCT group was increased significantly, and the expression of ADAM10 in the MCT+PNS group was decreased compared with the MCT group. These results indicate that PNS can improve the PAH induced by MCT in rats by inhibiting ADAM10/Notch3 signaling pathway.

The regulation of retinoid X receptor-mediated oxidative stress pathway in rat pulmonary ischemia/reperfusion injury / 生理学报

The aim of this study was to investigate the regulatory role of retinoid X receptor (RXR)-mediated oxidative stress pathway in rat pulmonary ischemia/reperfusion injury (PIRI) and the underlying mechanism. Seventy-seven male Sprague-Dawley (SD) rats were randomly divided into 7 groups (n = 11): control group, sham group, sham+9-cis-retinoid acid (9-cRA, RXR agonist) group, sham+HX531 (RXR inhibitor) group, ischemia/reperfusion (I/R) group, I/R+9-cRA group, and I/R+HX531 group. The unilateral lung I/R model was established by obstruction of left lung hilus for 30 min and reperfusion for 180 min in vivo. The rats in I/R+9-cRA and I/R+HX531 groups were given intraperitoneal injection of 9-cRA and HX531 before thoracotomy. After reperfusion, the left lung tissue was taken to evaluate the lung tissue injury, and the oxidative stress-related indexes of the lung tissue were detected by the corresponding kits. The lung tissue morphology and the ultrastructure of the alveolar epithelial cells were observed by HE staining and transmission electron microscope, respectively. The protein expression of RXR in lung tissue was observed by immunofluorescence labeling method, and the expression level of nuclear factor E2-related factor (Nrf2) protein was detected by Western blot. The results showed that, compared with the sham group, the I/R group exhibited obviously injured lung tissue, decreased SOD activity, increased MDA content and MPO activity, and down-regulated expression level of Nrf2 protein. Compared with the I/R group, the I/R+9-cRA group showed alleviated lung tissue injury, increased activity of SOD, decreased MDA content and MPO activity, and up-regulated expression levels of RXR and Nrf2 protein. The above-mentioned improvement effects of 9-cRA were reversed by HX531 treatment. These results suggest that RXR activation can effectively protect the lung tissue against I/R injury, and the mechanism may involve the activation of Nrf2 signaling pathway, the enhancement of antioxidant level and the reduction of oxidative stress response.

Effects of excessive endoplasmic reticulum stress on lung ischemia/reperfusion induced myocardial injury in mice / 中国应用生理学杂志

OBJECTIVE@#To investigate the effects of excessive endoplasmic reticulum stress on lung ischemia/reperfusion (I/R) induced myocardial injury in mice.@*METHODS@#Forty healthy SPF male C57BL/6J mice were divided into 4 groups randomly (=10):sham operation group (Sham group), lung I/R group (I/R group), endoplasmic reticulum stress (ERS) pathway agonist Tunicamycin group (TM) and ERS inhibitor 4-phenyl butyric acid group (4-PBA). The model of lung I/R injury was established by clamping the left hilum of lung for 30 min followed by 180 min of reperfusion. In sham group, only sternotomy was performed, the hilum of lung was not clamped, and the mice were mechanically ventilated for 210 min. In TM and 4-PBA groups, TM 1mg/kg and 4-PBA 400 mg/kg were injected intraperitoneally, respectively, at 30 min before establishment of the model. At 180 min of reperfusion, blood samples were collected from the orbit for determination of myocardial enzyme. The animals were then sacrificed, and hearts were removed for determination of light microscope, TUNEL, Caspase 3 enzymatic activity, real-time polymerase chain reaction and Western blot.@*RESULTS@#Compared with sham group, the cardiomyocytes had obvious damage under light microscope, and the serum creatine kinase-MB (CK-MB) and lactic dehydrogenase (LDH) activities, apoptosis index and Caspase 3 enzymatic activity were increased significantly, the expressions of p-Jun N-terminalkinase(p-JNK), Caspase 12, CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose regulated protein 78(GRP78) protein and mRNA were up-regulated in I/R, TM and 4-PBA groups (<0.01). Compared with I/R group, the cardiomyocytes damage was obvious under light microscope, and the serum CK-MB and LDH activities, apoptosis index and Caspase 3 enzymatic activity were increased significantly, the expressions of p-JNK, Caspase 12, CHOP and GRP78 protein and mRNA were up-regulated in group TM; while all above changes were relieved in group 4-PBA (<0.01). Compared with TM group, the cardiomyocytes damage was relieved under light microscope, and the serum CK-MB and LDH activities, apoptosis index and Caspase 3 enzymatic activity were decreased significantly, the expressions of p-JNK, Caspase 12,CHOP and GRP78 protein and mRNA were down-regulated in group 4-PBA.@*CONCLUSIONS@#The excessive endoplasmic reticulum stress participates in myocardial injury induced by lung ischemia/reperfusion (I/R) and inhibit excessive endoplasmic reticulum stress response can relieved myocardial injury.

Effect of dexmedetomidine on apoptosis and CHOP in hypoxia/reoxygenation injury A549 cell / 中国应用生理学杂志

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 (=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 (<0. 01). The AI value and expression of CHOP, Grp78, caspase-3 proteins and CHOP, Grp78 mRNA were significantly increased (<0.01). Compared with H group, the cell damage in HD group was decreased, the absorbance value increased (<0.01), the number of apoptosis cells decreased relatively (<0.01), the expression of CHOP, caspase-3 protein and CHOP mRNA decreased (<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 effect of Yiqi Wenyang Huoxue Huatan Fang on hypoxia-hypercarbia induced pulmonary hypertension and its mechanism / 中国应用生理学杂志

OBJECTIVE@#To investigate the effect of Yiqi Wenyang Huoxue Huatan Fang (YWHHF) on alleviating hypoxia-hypercarbia pulmonary hypertension by inhibiting endothelial-mesenchymal transition (EndoMT) BMP-7/Smads pathway.@*METHODS@#Fifty male healthy SD rats of clean grede, weighting (180~220) g, were randomly divided into 5 groups (=10):normoxia group (N), hypoxia-hypercarbia group (HH); YWHHF high dose group (YH), middle dose group (YM) and low dose group (YL). The rats in N group were kept in normal oxygen environment, the remaining four groups were intermittently exposed to hypoxia-hypercarbia environment (9%~11% O, 5%~6% CO) for 4 weeks, 6 days a week, 8 hours per day. The rats in YH, YM, YL groups were received YWHHF gavage in a dosageof 0.6, 0.3, 0.15g/kg respectively (3 ml/kg),the rats in N and HH groups were received equal volume of normal saline. After 4 weeks, the mean pulmonary arterial pressure(mPAP) was detected,the right ventricular free wall and left ventricle plus ventricular septum were isolated to determine the right ventricular hypertrophy index. Lung ultrastructural changes were surveyed under an electronic microscopy, the changes of pulmonary artery structure surveyed by immunofluorescence, the mRNA levels of alpha-smooth muscle actin (α-SMA)、platelet endothelial cell adhesion molecule-1 (CD31)、bone morphogenetic protein-7 (BMP-7)、drosophila mothers against decapentaplegic protein1/5/8 (Smad1/5/8) were detected by RT-PCR, and the protein levels of α-SMA、CD31、BMP-7、p-Smad1/5/8 and Smad1/5/8 were detected by Western blot.@*RESULTS@#Compared with N group, mPAP and the right ventricular hypertrophy index were increased,some significant injuries also were discovered under microscopic observation,the mRNA and protein expression of α-SMA was increased, and the mRNA expressions of CD31、BMP-7、Smad1/5/8 were decreased in the other four groups, the protein expressions of CD31、BMP-7、p-Smad1/5/8 were decreased(<0.05). Compared with HH group, the above changes in YH、YM、YL groups were all improved (<0.05).@*CONCLUSIONS@#YWHHF can inhibit EndoMT to alleviate pulmonary hypertension, and the mechanism may be related to the promotion of the expression of BMP-7/Smads pathway.

The role of endoplasmic reticulum stress in pulmonary hypertension in rat induced by chronic hypoxia and hypercapnia / 中国应用生理学杂志

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 (<0.01), and the ratio of pulmonary artery luminal area to total tube area (LA/TA) were decreased (<0.01), pulmonary artery smooth muscle cell apoptosis index were decreased (<0.05 or <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 ( <0.01), LA/TA and pulmonary artery smooth muscle cell apoptosis index were increased (<0.01, <0.05).The expressions of ERS related protein and mRNA were all decreased (<0.05 or <0.01).③Compared with the HH group, the mPAP, [RV/(LV+S)]and WA/TA of TM group were increased (<0.05 or <0.01), pulmonary artery middle layer thickened, LA/TA and pulmonary artery smooth muscle cell apoptotic index were decreased (<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.

Role of EndoMT in HHPH based on TGF-β/Smads signaling pathway and regulated by Yiqi-Wenyang-Huoxue-Huatan formula / 中国病理生理杂志

AIM:To investigate the relationship between transforming growth factor-β(TGF-β)/Smads signa-ling pathway and pulmonary arterial endothelial-mesenchymal transition(EndoMT)in hypoxia-hypercapnia pulmonary hy-pertension(HHPH)process and the regulatory effect of Yiqi-Wenyang-Huoxue-Huatan formula(YWHHF).METHODS:Healthy male SD rats were randomly divided into 5 groups:normal control(N)group,hypoxia-hypercapnia(HH)group, high-dose YWHHF(YH)group,middle-dose YWHHF(YM)group and low-dose YWHHF(YL)group.The rats in N group was housed in normoxic environment,and the rats in the other 4 groups were housed in hypoxia-hypercapnia environ-ment(9%~11%O2and 5%~6%CO2)for 4 weeks,8 h/d,6 d/week.The excess water vapor was absorbed by anhy-drous CaCl2,and CO2was absorbed by sodium hydroxide.The rats in YWHHF groups were put into the oxygen chamber before the same volume of YWHHF at different concentrations were given(200 g/L for YH group,100 g/L for YM group and 50 g/L for YL group).The average pulmonary artery pressure and the average carotid artery pressure were measured during the operation.After operation,the right ventricular free wall and left ventricle plus interventricular septum were col -lected for determining the right ventricular hypertrophy index.Moreover,the morphological changes of the lung tissues were observed under light microscope.The mRNA and protein levels of α-smooth muscle actin(α-SMA),CD31,TGF-β1 and Smad2/3,and the protein level of p-Smad2/3 were detected by RT-PCR and Western blot.RESULTS:Compared with N group,the pulmonary artery mean pressure,the mRNA and protein expression of α-SMA,TGF-β1 and Smad2/3,and the protein level of p-Smad2/3 were increased, the levels of CD31 were decreased(P<0.05), and the lung tissue damage was observed in the other 4 groups.Compared with HH group,the pulmonary artery mean pressure,the mRNA and protein expression of α-SMA,TGF-β1 and Smad2/3,and the protein level of p-Smad2/3 were decreased, while the mRNA and protein levels of CD31 were increased.Moreover,the lung tissue damage was reduced in YH,YM and YL groups.CON-CLUSION:TGF-β/Smads pathway may be involved in the process of EndoMT under hypoxia and hypercapnia condition, and YWHHF may reduce EndoMT by inhibiting the expression of TGF-β/Smads pathway-related molecules.

Effect of hydrogen molecule on apoptosis-related proteins and Nrf2 signa-ling pathway in glomerular mesangial cells cultured with high glucose / 中国病理生理杂志

AIM:To investigate the role of hydrogen molecule on apoptosis-related proteins in glomerular me-sangial cells cultured with high glucose and to explore its possible mechanism.METHODS:Mouse glomerular mesangial cells cultured in vitro were divided into 4 groups:normal control group(C group,5.5 mmol/L glucose),mannitol group(G group,5.5 mmol/L glucose+19.5 mmol/L mannitol),high glucose group(H group,25 mmol/L glucose),high glu-cose+hydrogen-rich water group(HH group,25 mmol/L glucose+hydrogen-rich water),and cultured for 48 h.The pro-tein levels of Bax,Bcl-2,cleaved caspase-3,nuclear factor E2-related factor-2(Nrf2),heme oxygenase-1(HO-1)and NAD(P)H:quinone oxidoreductase-1(NQO-1)were determined by Western blot ,and the mRNA expression of HO-1 and NQO-1 was determined by RT-PCR.The level of intracellular reactive oxygen species(ROS)was detected by dihydro-ethidium method,and the activity of superoxide dismutase(SOD)was measured by WST-8 assay.RESULTS:Compared with C group,the protein levels of Bax and cleaved caspase-3 were up-regulated,and Bcl-2 was down-regulated in H group(P <0.05).No significantly difference of the protein levels mentioned above between C and HH group was observed. Compared with H group,the protein levels of Bax and cleaved caspase-3 were down-regulated,and Bcl-2 was up-regulated in HH group(P <0.05).The level of intracellular ROS was higher and the activity of SOD was lower in H group than those in C group(P<0.05).However,there was no difference of the SOD activity between C group and HH group.The level of intracellular ROS decreased and the activity of SOD increased in HH group as compared with H group(P<0.05). Compared with C group,clearly reduced protein expression of Nrf2,HO-1 and NQO-1,and decreased mRNA expression of HO-1 and NQO-1 in H group were observed(P<0.05).Compared with H group,the protein levels of Nrf2,HO-1 and NQO-1 as well as the mRNA levels of HO-1 and NQO-1 were obviously increased in HH group(P<0.05 ).CONCLU-SION:Hydrogen molecule inhibits the expression of pro-apoptotic proteins and induces the expression of anti-apoptotic pro-teins in glomerular mesangial cells cultured with high glucose.The mechanism may be related to activation of Nrf 2 signaling pathway.

Role of TRPC6 in pulmonary artery smooth muscle cells proliferation and apoptosis under hypoxia and hypercapnia / 生理学报

The present study was to investigate the role of TRPC6 in pulmonary artery smooth muscle cells (PASMCs) proliferation and apoptosis under hypoxia and hypercapnia. PASMCs were isolated from chloral hydrate-anesthetized male Sprague-Dawley (SD) rats. Cellular purity was assessed by immunofluorescence staining for smooth muscle α-actin under fluorescence microscopy. Passage 4-6 PASMCs were starved for 24 h in serum-free DMEM and divided into 5 groups randomly: normoxia, hypoxia and hypercapnia, DMSO, TRPC6 inhibitor SKF-96365 and TRPC6 activator OAG groups. The normoxic group was incubated under normoxia (5% CO, 21% O, 37 °C) for 24 h, and the others were incubated with corresponding drugs under hypoxic and hypercapnic (6% CO, 5% O, 37 °C) atmosphere for 24 h. TRPC6 mRNA was detected by reverse transcription-PCR. TRPC6 protein was detected by Western blotting. The proliferation of PASMCs was performed by CCK-8 kit. Apoptosis of the PASMCs was detected using TUNEL assay. The [Ca]in the PASMCs was measured using Fura 2-AM fluorescence. The results showed that the expressions of TRPC6 mRNA and protein, and [Ca]were upregulated under hypoxic and hypercapnic conditions. Hypoxia and hypercapnia promoted cellular proliferation and inhibited apoptosis in the PASMCs. OAG enhanced the above-mentioned effects of hypoxia and hypercapnia, whereas SKF-96365 reversed these effects. These results suggest that TRPC6 may play a role in PASMCs proliferation and apoptosis under hypoxia and hypercapnia by regulating [Ca].

Excessive endoplasmic reticulum stress mediates brain damage in hypoxia hypercapnia induced pulmonary hypertension rats / 生理学报

The purpose of the present study was to investigate the effect of excessive endoplasmic reticulum stress (ERS) on the brain damage in hypoxia hypercapnia induced pulmonary hypertension (HHPH) rats. Forty healthy SPF male SD rats were randomly divided into four groups (n = 10 for each): control group, hypoxia hypercapnia group, ERS pathway agonist tunicamycin (TM) group and ERS pathway inhibitor 4-phenylbutyric acid (4-PBA) group. The rats of control group lived in normal environment, while the rats of other three groups were raised for four weeks in the tank with 8.5%-11% Oand 5%-6% CO. TM (0.08 mg/kg, twice a week) and 4-PBA (80 mg/kg, daily) were respectively intraperitoneally injected into the rats of TM and 4-PBA groups, and the hypoxia hypercapnia group was given the same volume of normal saline. The mean pulmonary artery pressure and heart perfusion of the rats were determined and recorded after four-week raising. Then the brain tissue of the rats were quickly taken out for the brain water content measuring and morphological changes observing. The Caspase-3 activity and the apoptotic index of the brain cells were also determined. The protein and mRNA expressions of p-JNK, Caspase-12, CHOP and GRP78 in brain tissues were detected by Western blot and RT-PCR. The results showed that compared with the control group, the mean pulmonary artery pressure, brain water content and brain cells apoptotic index, Caspase-3 activity, the protein and mRNA levels of p-JNK, Caspase-12, CHOP and GRP78 were increased (P < 0.05), and the brain tissues of the rats were obviously damaged in the rats raised in the hypoxia hypercapnia environment; compared with hypoxia hypercapnia group, the mean pulmonary artery pressure, brain water content, brain apoptotic index and Caspase-3 activity, p-JNK, Caspase-12, CHOP, GRP78 protein and mRNA expressions in TM group were increased (P < 0.05), and the brain tissues of the rats were obviously damaged, while all above changes were relieved in 4-PBA group (P < 0.05). These results suggest that excessive ERS may participate in the brain injury induced by HHPH in rats and inhibition of excessive ERS can relieve the brain injury in the rats with HHPH.

Effects of dexmedetomidine on hypoxia/reoxygenation injury-induced cell apoptosis and caspase-12 expression in A549 cells / 生理学报

To investigate the effects of dexmedetomidine (DEX) on hypoxia/reoxygenation (H/R) injury-induced cell apoptosis and caspase-12 expression, A549 cells were randomly divided into 4 groups: control group, DEX group, H/R group and DEX+H/R group. Cells of control and DEX groups were cultured in the normoxic incubator for 30 h. Cells of H/R and DEX+ H/R groups were incubated in the anoxic cultivation for 6 h, followed by normoxic culture for 24 h, and DEX (1 nmol/L) was added into the culture medium in DEX and DEX+H/R groups. Morphological changes were observed under the inverted microscope. Cell viability was detected by CCK-8. The apoptosis index (AI) of A549 cells was detected by TUNEL method. The activity of caspase-3 enzyme in cells was detected by using caspase-3 kit. The expressions of GRP78, caspase-12 protein and mRNA were determined by Western blot and RT-PCR respectively. Compared with control group, the morphological changes of the cultured cells were observed: some of the cell fusion occurred and the shape of the cells was multilateral; the cell viability was decreased significantly (P < 0.01), the number of apoptotic cells and the AI value, caspase-3 activity, and the expressions of GRP78, caspase-12 protein/mRNA were significantly increased (P < 0.01) in H/R group. While the administration of DEX alleviated the H/R injury-induced cell damage, obviously increased the cell viability (P < 0.01), significantly decreased the increment of apoptotic cells and the AI value induced by H/R injury (P < 0.01), and also dramatically decreased the H/R injury-induced high level of caspase-3 activity (P < 0.01) as well as high expression of caspase-12 protein and mRNA (P < 0.01). Taken together, the results suggest that DEX can effectively protect A549 cells from the H/R injury, which may be mediated by down-regulating the expression of caspase-12 and inhibiting cell apoptosis.

Effect of panax notoginseng saponins injection on the p38MAPK pathway in lung tissue in a rat model of hypoxic pulmonary hypertension / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of panax notoginseng saponins (PNS) injection on pulmonary artery pressure and the expression of p38MAPK in lung tissue of rats subjected to chronic hypoxia.</p><p><b>METHODS</b>Thirty adult male Sprague Dawley rats were randomly divided into three groups (ten in each group): rats in control group were exposed to normoxic condition and the rats in hypoxia group and PNS group were subjected to 4-week hypoxia, and PNS injection (50 mg · kg(-1) · d(-1)) was administrated intraperitoneally at 30 min in the PNS group daily before the rats were kept in the hypoxic chamber, while rats in the other two groups received equal dose of normal saline instead. After chronic hypoxia, mean pulmonary artery pressure (mPAP) and mean carotid artery pressure (mCAP) were measured. The heart and lung tissues were harvested, and right ventricle (RV) and left ventricle plus ventricular septum (LV+S) were weighed to calculate the ratio of RV/(LV+S). The expression of p38MAPK mRNA was determined by reverse transcription-polymerase chain reaction, the quantity of phosphorylated p38MAPK (p-p38MAPK) in rat lung tissues and pulmonary arterioles was determined by Western blot and immunohistochemistry.</p><p><b>RESULTS</b>Compared with the control group, mPAP and the ratio of RV/(LV+S) in the hypoxia group were increased, the expression of p-p38MAPK in pulmonary arterioles and p38MAPK mRNA in the lung were higher (P<0.05). The changes of these parameters in the hypoxia group were significantly attenuated by PNS treatment (P<0.05).</p><p><b>CONCLUSION</b>PNS injection was shown to prevent hypoxic pulmonary hypertension at least partly by regulating p38MAPK pathway.</p>

Effect of ERK1/2 on rat pulmonary artery smooth muscle cells Kv1.5 channel in the process of hypoxia / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To explore the effect of ERK1/2 MAPK pathway on the expression of Kv1.5 channel, a voltage-gated potassium ion channel, in rat pulmonary artery smooth muscle cells (PASMCs) and its mechanisms during the process of hypoxia.</p><p><b>METHODS</b>The PASMCs derived from SD rats were cultivated primarily. The third to sixth generation of PASMCs were divided into 5 groups randomly: (1) Normal group (N); (2) Hypoxic group (H); (3) Demethy sulfoxide(DMSO) group (HD); (4) U0126 group (HU): 10 micromol/L U0126; (5) Anisomycin group (HA): 10 micromol/L anisomycin. There were three dishes of cells in each group. The cells in normal group were cultured in normoxic incubator (5% CO2, 37 degrees C), the cells in other groups were added to 0.05% DMSO in the hypoxic incubator (5% CO2, 2% O2, 37 degrees C), all cells were cultured for 60 h. RT-PCR and Western blot were used to detected the espressions of Kv1.5 mRNA and protein in PASMCs.</p><p><b>RESULTS</b>Compared with N group, the expressions of Kv1.5 mRNA and protein in H, HD and HA groups were reduced significantly (P < 0.05); Compared with H group and HD groups, Kv1.5 mRNA and protein expressions in HU group were increased sharply (P < 0.05). Compared with the HU group, Kv1.5 mRNA and protein expressions in HA groups were significantly lower (P < 0.05).</p><p><b>CONCLUSION</b>Low oxygen reduced Kv1.5 mRNA and protein expressions, U0126 could resistant the Kv1.5 channel lower expression caused by hypoxia. Anisomycin had no significant effect on Kv1.5 channel expression under hypoxia, but the expression of Kv1.5 was still significantly lower than the normal oxygen group. These data suggest that hypoxia may cause hypoxic pulmonary hypertension by interfering ERK1/2 signaling pathway to inhibit Kv1.5</p>

Dynamic changes of IL-1β in rat myocardium during hypoxia/ reoxygenation transition / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the expression profile of interleuki-1β (IL-1β) in rat myocardium at different time points during hypoxia/reoxygenation(H/R)transition.</p><p><b>METHODS</b>The isolated Langendorff perfused rat heart model was established.Forty SD rats were randomly divided into sham group (A group) and hypoxia/reoxygenation group (H/R group). The H/R group rats were subdivided into H/R 0.5 h group(B group), H/R 1 h group(C group), H/R 2 h group(D group)according to reoxygenation time. The left ventricular development pressure(LVDP), maximal rates of increase/decrease of the left ventricular pressure(±dp/dtmax) were continuously recorded. The concentration of interleukin-1β(IL-lβ) and creatine kinase-MB (CK-MB) in myocardium was measured by ELISA. The mRNA expression of IL-lβ in myocardium was determined by RT-PCR. Microstructure of myocardium was observed under light microscopy.</p><p><b>RESULTS</b>The value of LVDP and ±dp/dtmax in hypoxia/reoxygenation group rat were significantly lower than that in sham group(P < 0.05). The expression of IL-lβ and CK-MB at protein level and the expression of IL-1β at mRNA level in hypoxia /reoxygenation group were higher than that in sham group(P < 0. 05). There were significant differences of the above parameters among H/R 0.5 h, 1 h, 2 h group(P <0.05). The concentration of IL-1β and CK-MB, the mRNA expression of IL-1β were higher in H/R 2 h group than that of other groups(P < 0.05).</p><p><b>CONCLUSION</b>The high expression of IL-Iβ in myocardium after myocardial hypoxia /reoxygenation in rats might lead to. ischemia/reperfusion injury.</p>

Effect of Xuebijing Injection on TLR4-NF-κB-TNF-α pathway of rats' myocardial anoxia/reoxygenation / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the role of Xuebijing Injection (XBJI) in inhibiting inflammatory factors associated with anoxia/reoxygenation myocardial inflammatory response of rats.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Effect of curcumin on caspase-12 and apoptosis in pulmonary ischemia/reperfusion injury mice / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the effect of curcumin (CUR) on cycteinyl aspirate specific protease-12 (Caspase-12) and pneumocyte apoptosis in pulmonary ischemia/reperfusion (I/R) injury mice.</p><p><b>METHODS</b>The in vivo unilateral in situ pulmonary I/R injury mouse model was established in C57BL/6J mice. Sixty experimental mice were randomly divided into six groups by random digit table, i. e., the sham-operation group (Sham), the I/R group, the I/R + dimethyl sulfoxide group (I/R + DMSO), the I/R + low dose CUR pre-treated group (I/R + CUR-100), the I/R + middle dose CUR pre-treated group (I/R + CUR-150), the I/R + high dose CUR pre-treated group (I/R + CUR-200), 10 in each group. Mice were euthanized and their left lungs were excised. Wet lung weight to dry lung weight (W/D) and the total lung water content (TLW) were tested. The morphological changes of the lung tissue were observed and index of quantitative evaluation for alveolar damage (IQA) detected under light microscope. The ultra-microstructure of the lung tissue was observed under electron microscope. The mRNA and protein expression levels of Caspase-12 and glucose regulated protein (GRP78) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Apoptosis index (AI) of the lung tissue was determined by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method.</p><p><b>RESULTS</b>Compared with the Sham group, expression levels of Caspase-12, GRP78 mRNA and protein all significantly increased in the I/R group (P < 0.05); W/D, TLW, IQA, and AI were all notably higher (P < 0.05, P < 0.01); the morphological and ultrastructural injury of the lung tissue were notably observed in I/R group. Compared with the I/R + DMSO group, expression levels of GRP78 mRNA and protein were increasingly higher in the I/R + CUR-100 group, the I/R + CUR-150 group, and the I/R +CUR-200 group (P < 0.05), expression levels of Caspase-12 mRNA and protein were lower (P < 0.05); W/D, TLW, IQA, and AI also decreased (P < 0.05, P < 0.01); the morphological and ultrastructural injury of the lung tissue were gradually alleviated in the I/R + CUR groups.</p><p><b>CONCLUSION</b>CUR had better effect on the lung protection against I/R injury, which might be related to inhibition for pneumocyte apoptosis associated with Caspase-12 in excessive unfolded protein response (UPR).</p>

Effect of siRNA silencing the role of JNK gene in excessive endoplasmic reticulum stress on lung ischemia/reperfusion injury / 中国应用生理学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>I/R induces excessive ERS in lung tissue, in which JNK pathway participates in apoptosis, leading to lung tissue injury.</p>

Effect of Xuebijing injection on TLR4--NF-kappaB--IL-1beta pathway of myocardial hypoxia/reoxygenation in rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Effects of ischemic postconditioning on pneumocyte apoptosis after lung ischemia/reperfusion injury in rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effects of ischemic postconditioning (IPostC) on pneumocyte apoptosis after lung ischemia/reperfusion injury in rats.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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) .</p><p><b>CONCLUSION</b>IPostC may attenuate pneumocyte apoptosis in LIRI by up-regulating expression of Bcl-2/Bax ratio and by inhibiting oxidant generation and neutrophils filtration.</p>

The effect of niflumic acid in hypoxic hypercapnia pulmonary vasoconstriction / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effect of chloride channel blocker--niflumic acid (NFA) on the pathological process of hypoxia hypercapnia-induced pulmonary vasoconstriction in rats.</p><p><b>METHODS</b>We used the model of hypoxia hypercapnia-induced pulmonary vasoconstriction rats, and divided the second, third branch pulmonary artery rings randomly into four groups (n = 8): control group (N group), hypoxia hypercapnia group (H group), DMSO incubation group (HD group), niflumic acid group (NFA group). Under acute hypoxia hypercapnia conditions, we observed the effects of the three stages of hypoxia hypercapnia-induced pulmonary vasoconstriction (HHPV) incubated by NFA in the second, third brach pulmonary artery rings. At the same time, the values of rings' tension changings were recorded via the method of hypoxia hypercapnia conditions reactivity. And investigated the effect of NFA to HHPV.</p><p><b>RESULTS</b>(1) Under the hypoxia hypercapnia condition, we observed a biphasic pulmonary artery contractile (the phase I rapid contraction and vasodilation; the phase II sustained contraction) response in both the second and the third branch pulmonary artery rings compared with the control group (P < 0.05 , P < 0.01); (2) The second and third pulmonary artery rings incubated by NFA which phase II persistent vasoconstriction were significantly attenuated compared with the H group (P < 0.05 , P < 0.01).</p><p><b>CONCLUSION</b>The blocker of the chloride channels attenuates the second and third branch pulmonary artery rings constriction in rat, especially the phase II persistent vasoconstriction, so then have an antagonistic effect on HHPV.</p>