CDKN2B-AS1 mediates proliferation and migration of vascular smooth muscle cells induced by insulin.

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the intimal hyperplasia in type 2 diabetes mellitus (T2DM) patients after percutaneous coronary intervention. We aimed to investigate the role of lncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in VSMC proliferation and migration, as well as the underlying mechanism. T2DM model mice with carotid balloon injury were used in vivo and mouse aortic vascular smooth muscle cells (MOVAS) stimulated by insulin were used in vitro to assess the role of CDKN2B-AS1 in VSMC proliferation and migration following vascular injury in T2DM state. To investigate cell viability and migration, MTT assay and Transwell assay were conducted. To elucidate the underlying molecular mechanisms, the methylation-specific polymerase chain reaction, RNA immunoprecipitation, RNA-pull down, co-immunoprecipitation, and chromatin immunoprecipitation were performed. In vivo, CDKN2B-AS1 was up-regulated in common carotid artery tissues. In vitro, insulin treatment increased CDKN2B-AS1 level, enhanced MOVAS cell proliferation and migration, while the promoting effect was reversed by CDKN2B-AS1 knockdown. CDKN2B-AS1 forms a complex with enhancer of zeste homolog 2 (EZH2) and DNA methyltransferase (cytosine-5) 1 (DNMT1) to regulate smooth muscle 22 alpha (SM22α) methylation levels. In insulin-stimulated cells, SM22α knockdown abrogated the inhibitory effect of CDKN2B-AS1 knockdown on cell viability and migration. Injection of lentivirus-sh-CDKN2B-AS1 relieved intimal hyperplasia in T2DM mice with carotid balloon injury. Up-regulation of CDKN2B-AS1 induced by insulin promotes cell proliferation and migration by targeting SM22α through forming a complex with EZH2 and DNMT1, thereby aggravating the intimal hyperplasia after vascular injury in T2DM.

[Panax notoginseng saponins improve monocrotaline-induced pulmonary arterial hypertension in rats by inhibiting ADAM10/Notch3 signaling pathway].

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.

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.

[Role of autophagy in liver injury induced by lung ischemia/ reperfusion in rats].

Objective: To investigate the liver injury induced by lung ischemia / reperfusion(LI/R) and the role of autophagy in its prevention and treatment. Methods: The lung ischemia/reperfusion injury(LI/RI) model was prepared by anesthetizing the rats, cutting the trachea for mechanical ventilation, and using an arterial clamp to close the pulmonary hilum to simulate the ischemic process, and releasing the arterial clamp after 30 min to resume perfusion for 3 h. SD rats(n=24)were randomly divided into sham operation(sham)group,ischemia/reperfusion(I/R)group,solvent(DMSO)group and autophagy inhibitor (3-MA) group, 6 rats in each group. The rats were intraperitoneally injected with medicine before operation. After the rat LI/RI model was established,the rats were killed, and the lung wet/dry weight ratio was used to evaluate the success of modeling, the venous blood was collected to measure the contents of ALT and AST, and the liver tissues were collected. Light and electron microscopes were used to observed the liver tissues and cell shapes. The protein and mRNA expression levels of autophagy related proteins were determined by Western blot and RT-qPCR to suggest autophagy levels. Results: Compared with sham group, the lung wet/dry weight ratios in other groups were elevated, and the liver tissues of other groups were damaged significantly. Serum levels of AST and ALT were increased significantly and liver tissue damage was obvious, especially in I/R group. The light microscopy showed that the arrangement of hepatic cords was disordered or broken, hepatic sinuses were dilated, and edema of liver cells were observed; transmission electron microscopy showed varying degrees of mitochondria swelling up in liver cells in the other groups. At the same time, the expressions of AMPK, Beclin 1 and LC3 mRNA were increased, but the expressions of mTOR and p62 mRNA were decreased; the protein expressions of p-AMPK, Beclin 1, LC3-B were increased significantly, but those of p-mTOR and p62 were decreased (all P＜0.05). Compared with DMSO group, the injury of liver tissue in 3-MA group was alleviated, the damage degree of mitochondrial ultrastructure was lower, the levels of AST and ALT were decreased, the transcription and protein expression levels of autophagy related protein in liver tissue were decreased (P＜0.05). However, the injury degree of IR and DMSO groups were similar, and there was no significant differences in each index (P＞0.05). Conclusion: Lung ischemia/reperfusion can cause liver injury in rats. Autophagy can mediate liver injury induced by lung ischemia / reperfusion in rats and inhibiting autophagy can effectively reduce liver injury induced by LI/R in rats.

[Effects of panax notoginseng saponins on pulmonary vascular remodeling in rats with pulmonary hypertension and its mechanisms].

Objective: To investigate the effects of panax notoginseng saponins (PNS) on pulmonary vascular remodeling and SIRT1/FOXO3a/p27 pathway in pulmonary arterial hypertension (PAH) rats. Methods: Male SD rats weighing 200~250g were randomly divided into control group, monocrotaline group (MCT) and monocrotaline + panax notoginseng saponins group (MCT+PNS), with 10 rats in each group. The rats in control group were injected intraperitoneally with normal saline 3 ml/kg on the first day, then injected intraperitoneally with normal saline 2.5 ml/kg every day. The rats in MCT group were injected intraperitoneally with MCT 60 mg/kg on the first day, followed by daily injection of normal saline 2.5 ml/kg. In MCT+PNS group, 60 mg/kg MCT was injected intraperitoneally on the first day, and 50 mg/kg PNS was injected intraperitoneally every day. The above models were fed conventionally for 4 weeks. After the modeling was completed, the mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP) of rats in each group were detected by right heart catheter method, weighed and calculated right ventricular hypertrophy index (RVHI), and the pulmonary vascular structure and morphological changes were observed by HE and Masson staining. The protein and gene expressions of SIRT1, FOXO3a, p27, PCNA and Caspase-3 were detected by qPCR and Western blot. Results: Compared with control group, mPAP, RVSP and RVHI in MCT group were increased significantly (P＜0.01), pulmonary vessels were thickened significantly and collagen fibers were increased, protein and gene expressions of SIRT1, FOXO3a, p27 and Caspase-3 were decreased (P＜0.05 or P＜0.01). The protein and gene expressions of PCNA were increased (P＜0.05). Compared with MCT group, the levels of mPAP, RVSP and RVHI in MCT+PNS group were decreased significantly (P＜0.05 or P＜0.01), pulmonary vascular thickening was alleviated and collagen fibers were reduced. The protein and gene expressions of SIRT1, FOXO3a, p27 and Caspase-3 were increased (P＜0.05 or P＜0.01), while the protein and gene expressions of PCNA were decreased (P＜0.05 or P＜0.01). Conclusion: Panax notoginseng saponins can relieve pulmonary vascular remodeling in rats with pulmonary hypertension by activating SIRT1/FOXO3a/p27 pathway.

M6A methylation-mediated elevation of SM22α inhibits the proliferation and migration of vascular smooth muscle cells and ameliorates intimal hyperplasia in type 2 diabetes mellitus.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) induced by insulin resistance facilitates intimal hyperplasia of type 2 diabetes mellitus (T2DM) and N6-methyladenosine (m6A) methylation modification mediates the VSMC proliferation. This study aimed to reveal the m6A methylation modification regulatory mechanism. In this study, m6A demethylase FTO was elevated in insulin-treated VSMCs and T2DM mice with intimal injury. Functionally, FTO knockdown elevated m6A methylation level and further restrained VSMC proliferation and migration induced by insulin. Mechanistically, FTO knockdown elevated Smooth muscle 22 alpha (SM22α) expression and m6A-binding protein IGF2BP2 enhanced SM22α mRNA stability by recognizing and binding to m6A methylation modified mRNA. In vivo studies confirmed that the elevated m6A modification level of SM22α mRNA mitigated intimal hyperplasia in T2DM mice. Conclusively, m6A methylation-mediated elevation of SM22α restrained VSMC proliferation and migration and ameliorated intimal hyperplasia in T2DM.

[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.

[The regulation of retinoid X receptor-mediated oxidative stress pathway in rat pulmonary ischemia/reperfusion injury].

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].

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 (n=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 (P<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 (P<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].

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 effect of Yiqi Wenyang Huoxue Huatan Fang on hypoxia-hypercarbia induced pulmonary hypertension and its mechanism].

OBJECTIVE: To investigate the effect of Yiqi Wenyang Huoxue Huatan Fang (YWHHF) on alleviating hypoxia-hypercarbia pulmonary hypertension by inhibiting endothelial-mesenchymal transition (EndoMT) via BMP-7/Smads pathway. METHODS: Fifty male healthy SD rats of clean grede, weighting (180~220) g, were randomly divided into 5 groups (n=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% O2, 5%~6% CO2) 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(P<0.05). Compared with HH group, the above changes in YH、YM、YL groups were all improved (P<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].

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.

[Excessive endoplasmic reticulum stress mediates brain damage in hypoxia hypercapnia induced pulmonary hypertension rats].

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% O2 and 5%-6% CO2. 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].

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.

[Role of TRPC6 in pulmonary artery smooth muscle cells proliferation and apoptosis under hypoxia and hypercapnia].

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% CO2, 21% O2, 37 °C) for 24 h, and the others were incubated with corresponding drugs under hypoxic and hypercapnic (6% CO2, 5% O2, 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 [Ca2+]i in the PASMCs was measured using Fura 2-AM fluorescence. The results showed that the expressions of TRPC6 mRNA and protein, and [Ca2+]i 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 [Ca2+]i.

[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.

[The effects of ERK1/2 pathway on the expression of calcium activated chloride channel in hypoxia in PASMCs rat model].

OBJECTIVE: To investigate the expression of mRNA and protein of Calcium activated chloride channel (CLCA2) in hypoxic pulmonary artery smooth muscle cell (PASMCs) of rat and it's relationship with ERK1/2 signal pathway. METHODS: PASMCs were randomly divided into 5 groups including normal group(N group), hypoxia group(H group), DMSO group(D group), U0126 group (U group) and Staurosporine aglycone group(SA group). The protein expression of CLCA2 in PASMCs was detected by Western blot.The mRNA expression of CLCA2 was detected by half quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The mRNA and protein expressions of CLCA2 in H group were significantly higher than N group (P<0.01). Comparing with D group,the mRNA and protein expressions of CLCA2 were significantly increased in U group (P<0.01),the mRNA expression of CLCA2 in SA group was obviously decreased (P<0.01) with slightly decreasing of its protein expression. CONCLUSIONS: Hypoxia promotes the expressions of mRNA and protein of CLCA2 in rat PASMCs. The ERK1/2 pathway activator Staurosporine aglycone reduces the mRNA and protein expression of CLCA2 in rats PASMCs and the ERK1/2 pathway inhibitor U0126 induces the upregulation of the mRNA and protein expressiosn of CLCA2 in rats PASMCs.

[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.

[The regulation of MAPK signaling pathway on cell proliferation and apoptosis in hypoxic PASMCs of rats].

OBJECTIVE: To explore the relationship between hypoxic pulmonary arterial smooth muscle cells(PASMCs)proliferation, apop-tosis and mitogen-activated protein kinases(MAPK) signal pathway in rats. METHODS: PASMCs were obtained from male SD rats by the enzyme digestion method and primarily cultured; PASMCs were identified through two methods:immunofluorescence staining and light microscopy; the 4~6th generation PASMCs of logarithmic growth state of good growth period were selected, and randomly divided into 7 groups:normoxic con-trol group (N), hypoxia group (H), DMSO group (D), extracellular signal-regulated kinase1/2(ERK1/2) inhibitor-U0126 group (U) and p38MAPK inhibitor-SB203580 group (S), the p38MAPK activator-Anisomycin group (A), the ERK1/2 activator-Staurosporine Aglycone group (SA). When all the models were completed, the all groups joined the CCK-8 to measure cell proliferation; cell apoptosis of each group was detected by TUNEL kit after the modeling. RESULTS: Compared with N group, the expression of OD value in H group was up-regulated (0.990 ±0.041 vs 1.143 ±0.033,P < 0.01). There was no statistical significance on PASMCs apoptosis index(AI) in H group (4.913 ±0.451 vs 5.452 ±0.557, P > 0.05); Compared With H group, there were no statistical significance on the expression of PASMCs OD value and apoptosis index(AI)in D group (1.143 ±0.033 vs 1.142 ±0.049,5.452 ±0.557 vs 5.402 ±0.651,P > 0.05); the expression of OD value in U group was down-regulated, and the expression of AI was up-regulated (1.143 ±0.033 vs 0.985 ±0.078, 5.452 ±0.557 vs 10.145 ±2.545, P < 0.01); the expression of OD value in S group was up-regulated, and the expression of AI was down-regulated (1.143 ±0.033 vs 1.295 ±0.039, 5.452 ±0.557 vs 3.093 ±0.409, P < 0.01); the expression of OD value in A group was down-regulated, and the expres-sion of AI was up-regulated (1.143 ±0.033 vs 0.347 ±0.067, 5.452 ±0.557 vs 25.753 ±1.262, P < 0.01); the expression of OD value in SA group was up-regulated, and the expression of AI was down-regulated (1.143 ±0.033 vs 1.685 ±0.100, 5.452 ±0.557 vs 1.700 ±0.095, P < 0.01). CONCLUSIONS: The regulation of PASMCs' proliferation and apoptosis under hypoxia condition have a relationship with the participation of MAPK signal pathway.