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.

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 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.

[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.

[Expression of KATP in pulmonary artery smooth muscle cells under hypoxia-hypercapnia condition and the relationship with p38 MAPK pathway].

The aim of the present study is to investigate the expressions of ATP-sensitive K(+) channels (KATP) in pulmonary artery smooth muscle cells (PASMCs) and the relationship with p38 MAPK signal pathway in rats. Male SD rat PASMCs were cultured in vitro, and a model of hypoxia and hypercapnia was reconstructed. PASMCs were divided to normal (N), hypoxia-hypercapnia (H), hypoxia-hypercapnia+DMSO incubation (HD), hypoxia-hypercapnia+SB203580 (inhibitor of p38 MAPK pathway) incubation (HS) and hypoxia-hypercapnia+Anisomycin (agonist of p38 MAPK pathway) incubation (HA) groups. Western blot was used to detect the protein expression of SUR2B and Kir6.1; semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of SUR2B and Kir6.1. The results demonstrated that: (1) Compared with N, H, HD and HS groups, the expressions of Kir6.1 mRNA and protein in PASMCs of HA group were decreased significantly (P < 0.01), but there were no differences among N, H, HD and HS groups (P > 0.05); (2) Compared with N group, the expressions of SUR2B mRNA and protein in H, HD, HS and HA groups were increased significantly (P < 0.05), but there were no differences among H, HD, HS and HA groups (P > 0.05). The results imply that: (1) Hypoxia-hypercapnia, SB203580 didn't change the expressions of Kir6.1 mRNA and protein in PASMCs, but Anisomycin decreased the expressions of Kir6.1 mRNA and protein, so Kir6.1 may be regulated by the other subfamily of MAPK pathway; (2) Hypoxia-hypercapnia raised SUR2B mRNA and protein expressions in PASMCs, but SB203580 and Anisomycin did not affect the changes, so the increasing of SUR2B mRNA and protein induced by hypoxia-hypercapnia may be not depend on p38 MAPK pathway.

[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 curcumin on caspase-12 and apoptosis in pulmonary ischemia/reperfusion injury mice].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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).

[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.

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.

[Effects of Panax notoginseng saponins on pneumocyte apoptosis and c-Jun N-terminal kinase in lung ischemia/reperfusion injury].

The aim of the present study is to investigate the effects of Panax notoginseng saponins (PNS) on pneumocyte apoptosis and apoptosis-related protein, as well as c-Jun N-terminal kinase (JNK) in lung ischemia/reperfusion (I/R) injury. Thirty Wistar rats were randomly divided into control group, I/R group and PNS group. The unilateral lung I/R model was replicated by obstruction of left lung hilus for 30 min and reperfusion for 120 min in vivo. The rats in PNS group were given intraperitoneal injection of PNS at 60 min before ischemia and 10 min before reperfusion. Some lung tissues sampled at the end of the experiment were assayed for wet/dry weight ratio (W/T). The expressions of phosphorylated JNK (p-JNK) and JNK protein were detected by Western blot. The expressions of Bcl-2, Bax and Caspase-3 protein were detected by immunocytochemistry techniques. The pneumocyte apoptotic index (AI) was detected by terminal deoxynuleotidy1 transferase mediated dUTP nick end labeling (TUNEL). The morphological and ultrastructure changes were observed under light microscope and electron microscope, and the injured alveolus rate (IAR) was counted as well. The results showed that compared to control group, I/R group showed increased expressions of p-JNK, Bcl-2, Bax and Caspase-3 protein (all P < 0.01), decreased ratio of Bcl-2/Bax (P < 0.05), and increased values of AI, W/T and IAR (all P < 0.01). Moreover, light microscope and electron microscope showed serious morphological and ultrastructure injury in I/R group. Compared to I/R group, PNS group showed markedly decreased expressions of p-JNK, Bax and Caspase-3 protein (all P < 0.01), increased expression of Bcl-2 protein and ratio of Bcl-2/Bax (both P < 0.01), and lower values of AI, W/T and IAR (all P < 0.01). Meanwhile, light morphological and ultrastructure injury was found to be alleviated in PNS group. These results suggest that PNS can protect lung tissue from I/R injury, and the mechanism may correlate with suppressing JNK signal pathway, up-regulating the ratio of Bcl-2/Bax which results in inhibition of Caspase-3 dependent apoptosis.

[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.

[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.

[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.

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.

[Involvement of metallothionein in the protection of lung ischemic preconditioning].

The aim of the present study was to investigate whether metallothionein was involved in the protection of lung ischemic preconditioning (IP) against lung ischemia-reperfusion (I/R) injury. Adult male Sprague-Dawley rats were randomly divided into 3 groups based upon the intervention (n=8): control group (C), lung I/R group (I/R), lung I/R+IP group (IP). At the end of the experiment, the content of metallothionein was tested in lung tissue. Blood specimens collected from the arteria carotis were tested for the contents of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and myeloperoxidase (MPO). The pneumocyte apoptosis index (AI) was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL). Ultrastructural changes of lung tissue were observed by using transmission electron microscope. The results showed that in I/R group, the content of metallothionein was decreased (P<0.05), the content of MDA and MPO activity were increased (P<0.01), and SOD activity was decreased (P<0.01), compared with those in control group. IP treatment significantly increased the content of metallothionein (P<0.01), attenuated the MDA level (P<0.05) and MPO activity (P<0.01), and improved SOD activity (P<0.01) in blood serum. The number of TUNEL-positive cells in IP group was significantly reduced compared with that in I/R group (P<0.01). There were abnormal ultrastructural changes in I/R group, which were markedly reversed in IP group. In conclusion, IP may protect lung against I/R injury by inducing the expression of metallothionein.

[Effects of lipid emulsion on hemodynamics and pharmacokinetics of mongrel dogs with bupivacaine-induced cardiac depression].

OBJECTIVE: To investigate the effects of Intralipid injection on hemodynamics and pharmacodynamics of mongrel dogs with bupivacaine-induced cardiac depression. METHODS: Fourteen dogs were randomly divided into Intralipid injection group (the experiment group) and normal saline group (the control group). 0.5% bupivacaine was administrated at a constant rate until the mean arterial pressure (MAP) decreased to 60% of the basic value. Intralipid injection was administered in the experiment group. The hemodynamic parameters were recorded and blood samples were taken from femoral artery at 0, 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, 240 min after stopping bupivacaine infusion for determination of plasma concentration by HPLC. The pharmacokinetic parameters were calculated by Drug and Statistics soft version 2.0. RESULTS: After the treatment of Intralipid injection, MAP, CO, HR increased significantly, compared to those of the control group (P < 0.05). The AUC(o-t) was (192 +/- 34)mg x L(-1) x min(-1) versus (271 +/- 81) mg x L(-1) x min(-1) and the AUC(0-infinity) was (231 +/- 62) mg x L(-1) x min(-1) versus (368 +/- 140) mg x L(-1) x min(-1) in the experiment group and the control group respectively. Vz (4.5 +/- 1.1 L/kg versus 3.3 +/- 0.9 L/kg) of bupivacaine in the experiment group was significantly higher than that in the control group (P < 0.05). CONCLUSION: Intravenous infusion of Intralipid have beneficial effects on the hemodynamics of mongrel dogs with bupivacaine-induced cardiac depression. One of the mechanism of the beneficial effects was probably that Intralipid injection increases the apparent volume of bupivacaine distribution and that decreases blood concentration. [Key words]