Effects of ketamine on α-synuclein and astrocyte in mice with Parkinson disease induced by MPTP

AIM To investigate the effects of ketamine at subanesthetic dose on α-synuclein and astrocyte in mice with Parkinson disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). METHODS Thirty-six healthy male C57BL/6 mice were randomly divided into 3 groups (12 mice per group): NaCl group (intraperitoneal injection of saline), MPTP group (intraperitoneal injection of MPTP) and ketamine group (intraperitoneal injection of MPTP and ketamine). The behavioral differences among the mice in the 3 groups were examined by tail suspension test and gait analysis test. Immunofluorescence staining and Western blot were used to detect the expression of α-synuclein and glial fibrillary acidic protein (GFAP) in the substantia nigra (SN), caudate putamen (CP) and visual cortex (CX). RESULTS According to the results of tail suspension test and gait analysis test, the mice in MPTP group showed increased duration of immobility and shortened step length compared with NaCl group, while those in ketamine group showed decreased duration of immobility and expanded step length compared with MPTP group (P<0.05). According to the results of immunofluorescence staining and Western blot, the mice in MPTP group showed significantly increased expression of α-synuclein and GFAP in SN, CP and CX compared with NaCl group, while those in ketamine group showed significantly decreased expression of a-synuclein and GFAP in SN, CP and CX compared with MPTP group (P<0.05). CONCLUSION Ketamine at subanesthetic dose inhibits the expression of α-synuclein and the proliferation of astrocytes in MPTP-induced PD mice.     

Expression of kinesin superfamily genes in striatum and substantia nigra in MPTP-treated mice

AIM: To investigated the effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the expression of kinesin superfamily (KIF) genes in striatum and substantia nigra in C57BL mice. METHODS: The Parkinson's disease model was established by consecutive administration of MPTP to C57BL mice. The levels of mRNA for five kinesin superfamily genes, KIF1A, KIF2, KIF3A, KIF4, and KIF5A, in striatum and substantia nigra of mice, were estimated by RT-PCR. RESULTS: In substantia nigra, the expression of KIF genes were decreased after MPTP treatment except KIF2 that showed no significant change. However, the expression of KIF1A, KIF3A and KIF4 were increased in striatum after MPTP treatment, while the expression of KIF2 and KIF5A were similar to that in substantia nigra. CONCLUSION: The lose of dopaminergic neurons in nigrostriatal pathway after MPTP treatment may be related to the expression of KIF genes.     

草莓CIPK基因家族的鉴定与表达分析

从拟南芥数据库中获得CIPK基因家族注册号,运用生物信息学分析的方法,在蔷薇科森林草莓（Fragaria vesca）数据库中得到CIPK基因家族成员19个,可分为6个亚族。该基因家族分布在草莓7条染色体中的6条上。其编码蛋白的氨基酸数157 ~ 1 196,理论等电点3.91 ~ 9.34,分子量18 667.68 ~ 133 714.31 D。基因结构分析表明,有11条基因只有1个外显子,其余基因外显子数2 ~ 15。亚细胞定位结果表明,该基因家族成员主要在细胞质、细胞核和叶绿体上表达。蛋白质二级结构预测表明,该基因家族成员主要以α–螺旋、β–转角和不规则卷曲为主。对上游2 kb区域启动子顺式作用元件分析表明,该基因家族成员对逆境胁迫应答MYB响应明显,除FvCIPK02、FvCIPK15、FvCIPK17外,其他基因均对脱落酸应答元件ABRE响应明显。qRT-PCR数据分析表明,FvCIPK16、FvCIPK10和FvCIPK09分别在PEG、ABA和NaCl处理下,草莓试管苗中的相对表达量最高,分别是对照的18.4倍、29倍、13倍,说明FvCIPK16强响应干旱胁迫,FvCIPK10强响应ABA诱导,FvCIPK09强响应高盐胁迫;另外发现各处理的FvCIPK03相对表达量均下调,推测FvCIPK03在植物逆境胁迫中起负调节作用。     

Activation of Nrf2 by 18α-GA affects proliferation of adult neural stem cells

AIM: To investigate the effect of nuclear factor E2-related factor 2 (Nrf2) activation by 18α-glycyrrhetinic acid (18α-GA) on the proliferation and self-renewal of adult neural stem cells (aNSCs), and to explore an effective way of maintaining the viability of aNSCs. METHODS: NSCs were dissociated from subventricular zone of the mice at postnatal days 0, 60, and 300. The expression levels of Nrf2 in the NSCs at various ages were compared. After treatment with 18α-GA, the expression of Nrf2 was examined by real-time PCR and Western blot. shRNA lentiviral vector (LV) carrying green fluorescent protein (GFP) gene was constructed to knock down Nrf2 expression. The knockdown efficiency in the aNSCs was detected by real-time PCR and Western blot. Subsequently, the aNSCs were divided into DMSO group, 18α-GA group, LV-GFP group and LV-Nrf2-shRNA group. BrdU incorporation assay, Tuj1 staining, CCK-8 assay, Hoechst 33342/PI staining and detection of reactive oxygen species (ROS) were performed to analyze the proliferation, differentiation, viability, apoptosis and oxidative stress levels of the NSCs. RESULTS: The mRNA expression level of Nrf2 in adult and aged NSCs was significantly lower than that in newborn NSCs (P<0.01), while the ROS level of aNSCs was significantly higher (P<0.05). After treatment with 18α-GA, the expression level of Nrf2 in the aNSCs was significantly up-regulated as compared with DMSO group (P<0.01). Increased number of BrdU⁺ and Tuj1⁺ cells was observed in 18α-GA group, indicating that 18α-GA-treated cells had higher viability (P<0.05). Meanwhile, there were fewer apoptotic cells and lower ROS level in 18α-GA group than those in DMSO group (P<0.05). After knockdown of Nrf2 in aNSCs and then treated with 18α-GA, there were less BrdU⁺ and Tuj1⁺ cells, as well as the aNSCs with lower viability in LV-Nrf2-shRNA group (P<0.05). Moreover, the ROS level was increased in LV-Nrf2-shRNA group as compared with LV-GFP group (P<0.05). CONCLUSION: Activation of Nrf2 by 18α-GA elevates the antioxidant capacity of aNSCs, thus ameliorating the cell proliferation and differentiation potentials.     

Upregulation of proteasome activity by 18α-GA promotes proliferation of late-passage BMSCs in vitro

AIM: To investigate the effect of 18 alpha-glycyrrhetinic acid (18α-GA) on delaying the senescent progress and promoting the proliferation in late-passage bone marrow mesenchymal stem cells (BMSCs). METHODS: Late-passage BMSCs were incubated with 2.0 mg /L 18α-GA or the same volume of DMSO for 30 d, and the cells were harvested to determine the proteasome activity. The expression of senescence-related proteins p53, p21 and p16 was detected by senescence-associated β-galactosidase (SA-β-Gal) staining and Western blot. The cell proliferation, the expression level of cell cycle-related proteins and cell cycle distribution of the cells were measured by CCK-8 assay, BrdU incorporation, Western blot and flow cytometry. RESULTS: Compared with DMSO group, the proteasome activity in 18α-GA group increased significantly by about 0.2 times (P<0.01). SA-β-Gal-positive cells in 18α-GA group decreased, and cell staining was lighter. The contents of p53 and p21 in 18α-GA group were decreased (P<0.05). The results of CCK-8 assay showed that the A value in 18α-GA group was 0.3 times higher than that in DMSO group (P<0.01). BrdU incorporation showed the increased proliferation in 18α-GA group compared with DMSO group (P<0.05). The cells in G₁ phase in 18α-GA group decreased significantly compared with DMSO group, while the cells in S phase increased significantly (P<0.05). The expression level of cyclin D1 in 18α-GA group was 2.8 times higher than that in DMSO group (P<0.01), and the CDK4 level was 1.4 times higher than that in DMSO group (P<0.05). CONCLUSION: Activation of the proteasome activity by 18α-GA delays the aging process in the BMSCs and promotes the cell proliferation via up-regulation of the cell cycle-related proteins.     

Effects of renal denervation on inflammatory factors in a rabbit model of early atherosclerosis

AIM: To evaluate the effects of renal denervation (RDN) on the expression of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and interleukin-6 (IL-6) in a rabbit model of early atherosclerosis. METHODS: New Zealand male rabbits were divided into control group, RDN+ high-fat diet (HFD) group (RDN group), sham+HFD group (sham group) and HFD group. The rabbits in later 3 groups were fed with 2% cholesterol for 8 weeks to establish an early atherosclerosis model. The blood samples were collected to test the levels of lipids, norepinephrine (NE), TNF-α, IL-1α and IL-6. The protein expression of angiotensin Ⅱ (Ang Ⅱ) was detected by the method of immunohistochemistry. The levels of nuclear factor-κB (NF-κB) and Ang II 1 type receptor (AT1R) were evaluated by Western blot. The mRNA expression of TNF-α, IL-1α and IL-6 was determined by real-time PCR. RESULTS: After 1 d of RDN procedure, the NE level was lower in RDN group than that in sham group (P<0.01). After 8 weeks, the NE level was lower in RDN group than that in sham group and HFD group (P<0.05), and triglyceride (TG) was lower in RDN group than that in HFD group (P<0.05). The protein expression of Ang II was decreased in RDN group compared with sham group and HFD group (P<0.01). The protein expression of NF-κB was lower in RDN group than that in sham group (P<0.05). The plasma levels of TNF-α and IL-1α were reduced in RDN group compared with sham group and HFD group (P<0.05). The mRNA expression of TNF-α, IL-1α and IL-6 was reduced in RDN group compared with sham group (P<0.05). CONCLUSION: RDN inhibits sympathetic activity, decreases the plasma level of TG, and alleviates inflammatory reactions in the rabbits with atherosclerosis.     

Effects of cordycepin on motor and cognition in Parkinson disease mice induced by MPTP

AIM:To investigate the effects of cordycepin on the motor and cognition in Parkinson disease mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). METHODS:C57BL/6 mice were intraperitoneally injected with MPTP at a dose of 30 mg/kg daily for consecutive 8 d to establish the model of Parkinson disease. HE staining was used to observe the cell number in the substantia nigra pars compacta (SNpc) from the mice. Western blot was used to detect the protein level of tyrosine hydroxylase (TH) in substantia nigra (SN). The effects of cordycepin on the motor, emotional change and cognitive behavior of the Parkinson disease mice were examined by open-field test (OFT), sponta-neous alternating behavior (SAB) and water maze test (WMT), respectively. RESULTS:Cordycepin significantly reduced the apoptosis of cells in SNpc and reversed the decrease in the expression of TH in SN induced by MPTP (P<0.05). Furthermore, cordycepin was able to improve the average speed in OFT (P<0.05), and increased the total number of arm entry and the accuracy in SAB (P<0.05), but had no obvious effect on the latency in WMT. CONCLUSION:Cordycepin is capable of attenuating the impairments of motor and explorative ability in the early stage of Parkinson disease mice, but does not alter the cognitive dysfunction.     

Over-expression of PGC-1α reverses mitochondrial function reduction and apoptosis in OGD/R-induced neurons

AIM: To investigate the effect of over-expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) on mitochondrial morphology and cell apoptosis in the cortical neurons with oxygen glucose deprivation/reoxygenation (OGD/R). METHODS: The whole gene sequence of PGC-1α was obtained from the cerebral cortex of C57BL/6 mice by RT-PCR and cloned into the eukaryotic expression vector pEGFP-N1. The pEGFP-N1-PGC-1α was identified by PCR, and transfected into cortical neurons. The level of PGC-1α expression was identified by Western blot. The cortical neurons transfected with pEGFP-N1 and pEGFP-N1-PGC-1α vectors were treated with OGD/R. The mitochondrial mass, reactive oxygen species (ROS) and ATP production, cell apoptosis and changes of cleaved caspase-3 were detected by MitoTracker Red staining, flow cytometry, ATP metabolic assay kit and TUNEL. RESULTS: Over-expression of PGC-1α inhibited the decrease in mitochondrial biogenesis capacity and the ROS formation of OGD/R neurons (P<0.05), enhanced the ability of ATP synthesis (P<0.01), inhibited neuronal apoptosis (P<0.01) and decreased the activation of caspase-3 (P<0.01). CONCLUSION: PGC-1α over-expression inhibits neuronal apoptosis with OGD/R treatment by promoting mitochondrial biogenesis, inhibiting the production of ROS and maintaining mitochondrial function. PGC-1α may be used as a target for the development of cerebral ischemia/reperfusion injury drugs.     

Effects of Aconiti tuber polysaccharides on hypercholesterolemia and hepatic cholesterol 7α-hydroxylase expression in rats

AIM: To investigate the effects of polysaccharides isolated from Aconiti tuber (Fuzi polysaccharides,FPS) on the prevention of hypercholesterolemia induced by high-cholesterol diet and the expression of hepatic cholesterol 7α-hydroxylase (cytochrome P450 7α-1, CYP7α-1) in rats.METHODS: Fifty male Wistar rats were randomly divided into 3 groups. The rats were fed with normal diet (control group), high-cholesterol diet (HC group) or high-cholesterol diet plus FPS (224, 448 or 896 mg·kg^-1·d^-1, FPS group) for 2 weeks. The serum lipid level, body weight, food-intake and fecal amount were measured at week 2. The pathological changes of the liver were observed with HE staining. The mRNA expression of hydroxy methylglutaryl coenzyme A (HMG-CoA) reductase and CYP7α-1, the protein level of CYP7α-1, and fecal bile acid were also detected at week 2.RESULTS: FPS significantly inhibited high-cholesterol diet-induced elevation of serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) (P<0.05). HE staining showed that FPS attenuated fatty degeneration in liver. Real-time PCR analysis showed that FPS significantly up-regulated the mRNA expression of CYP7α-1, but down-regulated the mRNA expression of HMG-CoA reductase (P<0.01). The protein level of CYP7α-1 was higher in FPS group than that in HC group (P<0.01). The level of fecal bile acid in HC-treated rats was higher than that in the control rats, and FPS stimulated the excretion of fecal bile acid (P<0.05).CONCLUSION: FPS significantly reduces serum cholesterol levels, which is associated with the up-regulation of hepatic CYP7α-1 expression and down-regulation of hepatic HMG-CoA reductase expression.     

Fluorocitrate inhibits regulatory effect of ischemic postconditioning on HIF-1α/iNOS signaling pathway after cerebral ischemia in tree shrews with brain injury

AIM: To investigate the regulatory effect of HIF-1α/iNOS signaling pathway on the neuroprotection of ischemic postconditioning (PC) in tree shrews, and to explore the mechanisms of deteriorated cerebral injury after inhibiting astrocyte (AS) metabolism. METHODS: Thrombotic cerebral ischemia was induced by photochemical reaction in tree shrews. Fluorocitrate (FC) was used to inhibit AS metabolism and the ischemic PC was established at 4 h after ischemia followed by clipped ipsilateral common carotid artery on the ischemia side for 3 times, 5 min/time. A total of 67 male tree shrews were randomly divided into 7 groups:control (n=9), ischemia (4 h and 24 h, n=9 for each group), ischemia with PC (4 h and 24 h, n=9 for each group), and FC pretreatment (4 h and 24 h, n=11 for each group). The cerebral infarction size was detected by TTC staining, and the histological changes of hippocampal neurons were observed under light microscope. The regional cerebral blood flow (rCBF) in ischemic cortex was monitored by laser Doppler brain flowmetry. The protein expression of iNOS in hippocampus was detected both by immunohistochemistry and Western blot. The production of NO detected by spectrophotometer. The level of HIF-1α in hippocampus analyzed by ELISA. RESULTS: The cerebral infarct volume was increased with prolonged duration of ischemia, and the changes of ischemia at 24 h were significant (P<0.05). The cortical rCBF was progressively decreased, and it was decreased at 4 h and 24 h after ischemia (P<0.05). The expression of HIF-1α and iNOS in hippocampus was enhanced, and the production of NO was increased significantly (P<0.05). Ischemic PC restored the cortical rCBF (P<0.05), reduced cerebral infarction volume (P<0.05), down-regulated iNOS expression and reduced NO production in the hippocampus (P<0.05). However, the cortical rCBF in FC pretreatment group was significantly lower than that in ischemic group (P<0.05), the neuronal damage was aggravated, and the infarction volume was increased after pretreatment with FC (P<0.05). CONCLUSION: Ischemic PC may reduce cerebral injury by regulating the expression of HIF-1α and iNOS. Inhibition of AS function may attenuate the protective effect mediated by ischemic PC and aggravate brain injury.     

Effect of AG490 on expression of VEGF and HIF-1α in HEL cells

AIM: To investigate the effect of AG490 on the expression of VEGF and HIF-1α, and the capacity of invasion in human erythroleukemia (HEL) cells. METHODS: The HEL cells were treated with AG490 at different concentrations. The cell viability was detected by CCK-8 assay. The apoptosis was detected by Hoechst staining. The apoptosis and the cell cycle were analyzed by flow cytometry. The capacity of migration was evaluated by Transwell assay. The mRNA expression level of JAK2 was measured by RT-PCR. The protein levels of p-JAK2, VEGF and HIF-1α were determined by Western blot. RESULTS: The HEL cell viabilities were 88%, 75%, 48%, 10% and 0.12% after treated with AG490 at 20, 40, 60, 80 and 100 μmol/L for 48 h, respectively. The results of Hoechst staining showed that brilliant blue cells in 80 μmol/L AG490 group was significantly increased compared with control group for 48 h. The apoptosis rate of 80 μmol/L AG490 group was significantly increased compared with control group at 48 h after AG490 treatment. The number of membrane-permeating HEL cells in 20 μmol/L AG490 group at 24 h after AG490 treatment was significantly lower than that in control group (P<0.05). The mRNA level of JAK2 decreased in a concentration-dependent manner after the HEL cells were treated with different concentrations of AG490 for 48 h. The protein levels of p-JAK2, VEGF and HIF-1α were lower in AG490 treatment groups than those in control group (P<0.05). CONCLUSION: AG490 inhibits the expression of VEGF and HIF-1α in HEL cells by inhibiting JAK2 pathway.     

Effects of MG132 on protein expression of SnoN and fibrosis-related indicators in NRK-52E cells after incubated with high concentration of glucose

AIM: To investigate the effects of proteasome inhibitor MG132 on the expression of SnoN in renal tubule epithelial cells incubated in high glucose, and to explore the possible mechanism and function that MG132 reduces or slows down renal tubular interstitial injury after incubated in high glucose. METHODS: The NRK-52E cells were divided into normal control group (NG), high glucose group (HG) and high glucose plus pretreatment with different doses of MG132 group (HG+MG132). The immunofluorescence staining was used to detect the protein expression of E-cadherin and α-smooth muscle actin (α-SMA) in NRK-52E cells under different conditions. The relative protein expression levels of SnoN, Smad ubiquitination regulatory factor 2 (Smurf2), Arkadia, E-cadherin, α-SMA and collagen type Ⅰ(Col-Ⅰ) were detected by Western blotting. RESULTS: Compared with NG group, the expression of E-cadherin and SnoN was decreased (P<0.05), while the expression of α-SMA, Col-Ⅰ, Smurf2 and Arkadia was increased (P<0.05). Compared with HG group, the protein expression of SnoN and E-cadherin was significantly up-regulated in HG+MG132 group (P<0.05), and the protein expression of α-SMA and Col-Ⅰ was significantly down-regulated in a dose-depended manner (P<0.05). However, no effect on the protein expression of Smurf2 and Arkadia was observed. CONCLUSION: MG132 inhibits the degradation of SnoN protein induced by high glucose, thus reducing the renal fibrosis.     

Effect of hypoxia-inducible factor-1α knockdown by siRNA on viability and CEACAM1 expression in oral squamous cell carcinoma

AIM: To investigate the relationship between hypoxia-inducible factor-1α (HIF-1α) and viability and apoptosis of oral squamous cell carcinoma and its mechanism. METHODS: The expression of HIF-1α and carcinoembryonic antigen-related cell adhesion molecular 1 (CEACAM1) at mRNA and protein levels in oral squamous cell carcinoma cell lines Tca8113 and CAL27 and normal epithelial cell line NOK was determined by RT-PCR and Western blot. The expression of HIF-1α in CAL27 cells was silenced by RNA interference (RNAi) technique. The cells were divided into blank control group, non-sense control group and siRNA-HIF-1α group. The viability of CAL27 cells was measured by MTT assay and the apoptotic rate was analyzed by flow cytometry. The protein levels of HIF-1α, P21, vascular endothelial growth factor (VEGF), Bcl-2 and Bax were examined by Western blot. RESULTS: The expression of HIF-1α and CEACAM1 in oral squamous cell carcinoma cells was significantly higher than that in normal cells (P<0.05), and the expression of HIF-1α and CEACAM1 was positively correlated. The protein expression of HIF-1α in siRNA-HIF-1α group was significantly lower than that in blank control group (P<0.05). Knockdown of HIF-1α significantly inhibited CAL27 cell viability (P<0.05), promoted apoptosis (P<0.05), increased the protein levels of P21 and Bax (P<0.05), and significantly decreased the levels of VEGF and Bcl-2 (P<0.05). CONCLUSION: HIF-1α is over-expressed in oral squamous cell carcinoma. Knockdown of HIF-1α significantly inhibits cell viability and promotes apoptosis possibly through regulating the expression of HIF-1α downstream target genes and tumor angiogenesis.     

Roles of gap junction in TGF-β1-induced proliferation of vascular smooth muscle cells from spontaneous hypertensive rats

AIM: To investigate whether gap junction participates in transforming growth factor β₁(TGF-β₁)-induced proliferation of spontaneous hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). METHODS: The thoracic aorta of the rats were sampled. The primary SHR VSMCs were isolated and cultured in vitro. The cells were divided into 4 groups: control group, TGF-β₁ group,18α-glycyrrhetinic acid(18α-GA) group and TGF-β₁+18α-GA group. The proliferation of SHR VSMCs was observed by the methods of MTT and flow cytometry. The protein expression and co-localization of connexin(Cx)43 and Cx40 in SHR VSMCs were detected by immunofluorescence staining. The protein levels of Cx43 and Cx40 in the cells were also measured by Western blotting. The method of molecular dye transfer (scrape dye transfer method) was applied to detect the function of gap junction in SHR VSMCs. RESULTS: The protein expression of Cx43 and Cx40 in SHR VSMCs was positive and co-localized in the cytoplasm. Compared with control group, the percentage of S-phase detected by cell cycle and A value detected by MTT in TGF-β₁ group were obviously increased (P<0.05), indicating that the proliferation of the cells was enhanced. However, the proliferation of the cells decreased in 18α-GA group (P<0.05). Compared with TGF-β₁ group, the percentage of S-phase and A value in TGF-β₁+18α-GA group were both significantly decreased (P<0.05), indicating that the proliferation of the cells decreased. Compared with control group, the protein expression of Cx43 in TGF-β₁ group was increased (P<0.05), whereas the protein expression of Cx40 was not changed (P>0.05), and the protein expression of Cx43 and Cx40 in 18α-GA group were decreased (P<0.05). Compared with TGF-β₁ group, the expression of Cx43 in TGF-β₁+18α-GA group was significantly decreased (P<0.05),but no difference of the Cx40 protein levels between the two groups was observed. Compared with control group, the function of gap junction detected by scrape dye transfer method in TGF-β₁ group was enhanced (P<0.05), and weakened in 18α-GA group (P<0.05). Compared with the TGF-β₁ group, the function of gap junction in TGF-β₁+18α-GA group was significantly attenuated (P<0.05). CONCLUSION: TGF-β₁ enhances the function of gap junction to stimulate the proliferation of SHR VSMCs through the expression of Cx43 protein. The expression of Cx40 protein may not play a major role in this process.     

Expression of hypoxia-inducible factor 1α in human gingival tissues with chronic periodontitis

AIM：To observe the expression of hypoxia-inducible factor 1α (HIF-1α) in human gingival tissues with chronic periodontitis. METHODS：A total of 55 volunteers, including 15 healthy controls, 20 cases of moderate chronic periodontitis and 20 cases of severe chronic periodontitis, were involved in this study, and their gingival specimens were taken and fixed in 4% neutral formalin. The histological changes of gingival tissues were observed by HE staining, and the expression of HIF-1α in gingival tissues was detected by immunohistochemical staining. RESULTS： The proportion of HIF-1α positive cells in gingival tissues was significantly higher in chronic periodontitis groups than that in healthy control group (P<0.01), and that in severe chronic periodontitis group was significantly higher than that in moderate chronic periodontitis group (P<0.05). There was a significantly positive correlation between the severity of chronic periodontitis and the proportion of HIF-1α positive cells in gingival tissues. CONCLUSION：The expression of HIF-1α in human gingival tissues is increased with the severity of chronic periodontitis, suggesting that hypoxia may play an important role in chronic periodontitis.     

Hypoxia-inducible factor-1α attenuates myocardial inflammatory injury induced by ischemia and reperfusion in rats

AIM:To investigate the role of hypoxia-inducible factor-1α (HIF-1α) stable expression in myocardial inflammatory injury induced by ischemia and reperfusion (I/R) in rats. METHODS:Male Wistar rats were randomly divided into 4 groups:sham operation (sham) group, I/R group, HIF-1α stabilizer dimethyloxalyl glycine (DMOG)+I/R group and HIF-1α inhibitor YC-1+I/R group. The protein expression of myocardial Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) was determined by Western blot. The mRNA levels of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, TLR4 and NF-κB were detected by real-time PCR. The myeloperoxidase (MPO) activity in the myocardial tissues was measured. HE staining was used to observe the infiltration of inflammatory cells. RESULTS:HIF-1α decreased the infiltration of inflammatory cells, the MPO activity, and the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the myocardial tissues. HIF-1α also reduced the expression of TLR4 and NF-κB at mRNA and protein levels (P<0.05). CONCLUSION:The stable expression of HIF-1α has an anti-inflammatory effect on the myocardial tissues after I/R injury in rats. The mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway.     

Effect of 27nt-miRNA on regulation of SM22α expression in vascular smooth muscle cells and its effect on cell viability,migration and phenotypic changes

AIM:To investigate the effect of 27nt-microRNA (27nt-miRNA) on the expression of smooth muscle 22α protein (SM22α) and the cell viability, migration and phenotypic changes of vascular smooth muscle cells (VSMCs). METHODS:The highly expression plasmids of 27nt-miRNA, and anti-27nt-miRNA and negative control plasmids were constructed, packaged with lentivirus and transfected into the rat primary VSMCs. Platelet-derived growth factor BB (PDGF-BB) was added to induce VSMCs phenotype conversion. The cell viability was measured by MTT assay. The migration ability was detected by scratch assay. The mRNA and protein expression of SM22α was determined by RT-PCR, immunocytochemical staining and Western blot. RESULTS:Compared with normal group, the cell viability in PDGF-BB group was increased (P<0.05), the migration ability was increased (P<0.05) and the expression of SM22α at mRNA and protein level was decreased (P<0.05). Compared with negative control lentiviral group, the cell viability in 27nt-miRNA over-expression group was decreased (P<0.05), the migration ability was decreased (P<0.05), and the mRNA and protein expression of SM22α was increased (P<0.05). While in anti-27nt-miRNA group, the cell viability was increased(P<0.05), the migration ability was increased (P<0.05), and the mRNA and protein expression of SM22α was decreased (P<0.05). CONCLUSION:27nt-miRNA significantly increases the expression of SM22α, while inhibits the viability and migration ability of VSMCs, and inhibits its phenotypic shift from contractile to synthetic.     

Protective effects of Zhenwu decoction on kidney injury of diabetic rats induced by streptozotocin

AIM：To investigate the effects of Zhenwu decoction (ZWD) on the expression of α-smooth muscle actin (α-SMA) and NF-κB in diabetic nephropathy (DN) rats. METHODS：Diabetic rat model was induced by intrape-ritoneal injection of streptozotocin (STZ), and the animals were randomly divided into STZ group (n=22) and STZ+ZWD group (n=23). The normal rats served as control (n=16). All rats were sacrificed on 8 weeks after modeling. Biochemical assay and pathological observation (HE staining and transmission electron microscopy) were used to evaluate the effects of Zhenwu decoction on the renal function and pathological morphology. The body weight, renal index, blood glucose, total urinary protein in 24 h, and superoxide dismutase (SOD), malondialdehyde (MDA),inducible nitric oxide synthase (iNOS) were determined as well. Western blotting was used to observe the effects of Zhenwu decoction on the expression of α-SMA and NF-κB in diabetic nephropathy (DN) rats. RESULTS：Compared with normal group, the renal index, blood glucose concentration, total urinary protein in 24 h, blood urea nitrogen (BUN), serum creatinine (SCr) and MDA were significantly higher and body weight was lower in DN rats (P<0. 05). Pathological examination of the kidneys in DN group showed glomerular hypertrophy, glomerular basement membrane thickening, tubular epithelial cell degeneration, mesangial matrix proliferation, protein cast formation in some renal tubules. The protein expression levels of α-SMA and NF-κB were markedly increased (P<0.05). After ZWD treatment, the level of renal index, total urinary protein in 24 h, BUN, SCr and the expression of α-SMA and NF-κB at the protein level were significantly decreased (P<0.05). The renal histological injury in ZWD group was significantly ameliorated. CONCLUSION：Zhenwu decoction might protect kidney against STZ-induced injury via decreasing the expression of α-SMA and NF-κB.     

H IF-1αm ediates effect of interm ittent hypoxia on biological behavior of A 549 cells in vitro

ATM: To investigate whether hypoxia-inducible factor 1α (HIF-1α) mediates the effect of intermittent hypoxia on A549 cell viability, apoptosis and invasive ability METHODS: A549 cells were transfected with HIF-1α-siRNA and cultured under intermittent hypoxia. The expression of HIF-1α and its downstream genes, such as Bcl-2, Bax, P53, P21 and VEGF at mRNA and protein levels was determined by real-time PCR and Western blot. The viability of the A549 cells was measured by MTT assay. The apoptosis and cell cycle distribution of the A549 cells were examined by flow cytometry. The invasive ability of the A549 cells was detected by transwell test. RESULTS: The expression levels of HIF-1α, Bcl-2 and VEGF in non-HIF-1α-siRNA transfected A549 cells cultured in intermittent hypoxia environment[blank controlgroup(IH C),empty vector control group (IH E) and negative control group (IH N)] were higher than those in the A549 cells in normoxia group (RA), but the expression levels of Bax and P21 were lower than those in RA group (P<0.05). The siRNA-mediated HIF-1α gene silencing[intermittent hypoxia silenced group (IHS)] resulted in obvious down-regulation of HIF-1α, Bcl-2 and VEGF, and significant increase in the protein expression of P21 and Bax(P<0.05). The expression level of P53 in intermittent hypoxia groups was significantly higher than that in RA group, and no significant difference of P53 expression in different intermittent hypoxia groups was observed. Compared with normoxia, intermittent hypoxia resulted in significantly enhanced cell viability, decreased apoptosis, and enhanced invasive ability of non-HIF-1α-siRNA transfected A549 cells (P<0.05). The siRNA-mediated HIF-1α gene silencing resulted in significant cell viability inhibition, elevated apoptotic rate and decreased invasive ability under hypoxic condition (P<0.05).CONCLUSION: Intermittent hypoxia promotes the viability and invasion of A549 cells by HIF-1α-mediated downstream gene expression. HIF-1α gene silencing inhibits A549 cell growth and invasion under intermittent hypoxia by inhibition of HIF-1α signal pathways in vitro.     

Effect of calcitonin gene-related peptide on myocardin expression and phenotypic switch in vascular smooth muscle cells

AIM: To investigate the effects of calcitonin gene-related peptide (CGRP) on myocardin expression and phenotypic switch in vascular smooth muscle cells (VSMCs). METHODS: VSMCs were obtained by aortic tissue adherent culture and treated with angiotensin Ⅱ (AngⅡ), AngⅡ + CGRP or AngⅡ + CGRP + CGRP_8-37. The protein expression of myocardin and the phenotypic proteins of the VSMCs was detected by Western blot. RESULTS: The expression of myocardin in cultured VSMCs showed downregulation along with time expansion. The protein level of myocardin was higher at 48 h and 72 h than that at baseline in the cultured VSMCs (P<0.05). However, the myocardin was lower at 48 h and 72 h than that at baseline after treatment with CGRP in cultured VSMCs (P<0.05). Furthermore, at 48 h in cultured VSMCs, the myocardin decreased along with α-smooth muscle actin (α-SMA) (P<0.05), and osteopontin (OPN) increased (P<0.05) in AngⅡ group compared with control group. After treatment with CGRP, the levels of myocardin and α-SMA become higher (P<0.05) but OPN was lower (P<0.05) in CGRP group than those in AngⅡ group. CGRP_8-37 abrogated CGRP-induced increase in myocardin and α-SMA and decrease in OPN in CGRP_8-37 group compared with CGRP group. CONCLUSION: CGRP may regulate the phenotypic switch of the VSMCs and maintain the cells in contractile phenotype through the upregulation of myocardin protein, which may be accomplished by the combination of CGRP and its receptor.