Combination of LPS and z-VAD-FMK mediates necroptosis in IFN-γ-pretreated macrophages

AIM:To explore the mechanism of necroptosis in M1 macrophages mediated by lipopolysaccharide (LPS) combined with z-VAD-FMK. METHODS:THP-1 cells were induced to differentiate into M0 and M1 macrophages with phorbol 12-myristate 13-acetate (PMA) and interferon-γ (IFN-γ). The release of lactate dehydrogenase (LDH) and TUNEL-positive cells at different time points after LPS (100 μg/L) treatment were detected, and the effects of different inhibitors were observed. The protein levels of receptor-interacting protein (RIP) 1, RIP3, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), P38 and NOD-like receptor protein 3 (NLRP3) were determined by Wes-tern blot. RESULTS:LPS mediated cell death, and its combination with z-VAD-FMK specifically mediated necroptosis in M1 macrophages rather than M0 ones. The expression of RIP3 and NLRP3 was upregulated by IFN-γ, and LPS-mediated phosphorylation of JNK was also enhanced by IFN-γ. The inhibitors against RIP3 and JNK partly blocked LDH release mediated by LPS combined with z-VAD-FMK. CONCLUSION:Combination of LPS and z-VAD-FMK mediates necroptosis in IFN-γ-pretreated macrophages possibly by upregulation of RIP3 and enhancement of LPS-mediated JNK phosphorylation.     

Role of NF-κB in inducing HEL cell proliferation and apoptosis during human cytomegalovirus infection

AIM:To investigate whether human cytomegalovirus(HCMV) regulate human embryonic lung fibroblast(HEL) cell proliferation and apoptosis by activating NF-κB.METHODS:Immunohistochemistry and Western blot analysis were used to detect the NF-κB translocation and/or Bcl-2 and the levels of I-κBα during HCMV infection. Apoptotic cell were examined by flow cytometry, and the HEL cell proliferation was determined by MTT.RESULTS:The levels of NF-κB in the nucleus reached highly 48 h postinfection, and the levels of I-κBα were low 24 h postinfection. The activity of NF-κB was inhibited 120 h postinfection. The levels ofbcl-2was accorded with the activity of NF-κB. HCMV promoted HEL cells to proliferate before 72 h postinfection and induced apoptosis 120 h postinfection.CONCLUSION:NF-κB plays a role in HEL cell proliferation and apoptosis during HCMV infection, and it involves in the pathological mechanisms of diseases associated with HCMV infection.     

Effects of β3 integrin on adhesion and migration of vascular smooth muscle cells induced by growth factor

AIM: To investigate the effect of platelet-derived growth factor (PDGF) on β₃ integrin gene expression and the role of β₃ integrin on adhesion, migration and proliferation of vascular smooth muscle cells (VSMC) induced by PDGF. METHODS: β₃ integxin gene expression was detected by RT-PCr. After β₃ integrin extracellular do-main was blocks, VSMC adhesio, migration and proliferation were measured by adhesion assay awound-culture model an [³H]-TSR incorporation respectively.RESULTS: After the interaction between β₃ integrin and extracellular matrix was blocked, VSMC proliferation was inhibited in some degree and the rate of [³H]-TdR incorporation into VSMC decreased 39%. The cell adhesion and migration were significantly inhibited when 10 mg/L anti-β₃ integrin antibody was added (P＜0.05). When VSMC were treated by PDGF for 6 hours, the expression of β₃ integrin gene was 87% higher than that of control. CONCLUSION: PDGF significantly induces expression of β₃ integrin gene in VSMC, and the interaction between β₃ integrin and ECM protein may play an important role in VSMC adhesion and migration.     

Effect of tenuigenin on tau protein phosphorylation at Ser396 site in neurons of AD rats induced by Aβ1-40

AIM:To investigate the effect of tenuigenin(TEN) on hyperphosphorylation of tau protein in neurons of amyloid β-peptide_1-40(Aβ_1-40) -induced Alzheimer disease(AD) rats. METHODS:Aβ_1-40 was injected into hippocampus CA1 region of the rats to establish AD model. TEN at different doses(18.5 mg/kg, 37.0 mg/kg and 74.0 mg/kg) was intragastrically administered. The protein expression of protein kinase A(PKA),protein phosphatase 2A(PP2A), total tau and p-tau(Ser³⁹⁶) in the neurons was observed by the method of immunohistochemistry. The protein content of total tau and p-tau(Ser³⁹⁶), and the expression level of PKA and PP-2A were detected by Western blotting analysis. RESULTS:In Aβ_1-40 group, the level of total tau, the phosphorylation of tau protein and the expression of PKA were significantly increased compared with those in sham operation group. Meanwhile, the expression of PP2A in Aβ_1-40 group was lower than that in sham operation group. In TEN treatment group, the level of total tau, the phosphorylation of tau protein and the expression of PKA were markedly decreased, and the expression of PP2A was increased as compared with Aβ_1-40 group. CONCLUSION:TEN may protect the neurons from the toxic effect of Aβ_1-40 and reduce the hyperphosphorylation of tau(Ser³⁹⁶) in the neurons of AD rats by activating the expression of PP2A and inhibiting the expression of PKA.     

大葱新品种郑研寒葱的选育

郑研寒葱是以日本宏太郎为母本，郑州长白条为父本，利用杂交育种技术，单株选择与系统选育相结合的方法培育
出的大葱新品种。株高95～120 cm，葱白长40～50 cm，横径2～3 cm，功能叶5～7 片，叶色浓绿，蜡粉多，单株鲜质量 0.25～0.65
kg，每 667 m² 产量 5 200 kg 以上；田间表现抗寒性强，抗紫斑病、霜霉病和锈病的能力强于对照章丘大葱，抗倒伏；葱白洁
白致密，辣味浓，有香味，风味佳；货架期长，耐贮藏。适合我国中原地区及西部、北部地区栽培。     

Protective effects of berberine in pancreatic islet beta cells

AIM: To explore the protective effects of berberine （Ber） on islet beta cells and related possible mechanisms. METHODS: The injury of INS-1 cells was induced by treatment with alloxan (Axn). Berverine was then given at serial concentrations. The cells were divided into control group (Con), injury group (Axn), low-dose berberine group (LBer), medium-dose berberine group (MBer) and high-dose berberine group (HBer). Flow cytometry was employed to detect the apoptosis. The activation of PTEN/PI3K/Akt and HNF-1α/PDX-1 pathways and the protein level of cleaved caspase-3 were evaluated by Western blotting. The insulin releases under normal or high-glucose stimulation were measured by ELISA. RESULTS: Compared with Con group, the apoptotic rate increased significantly in Axn group. Berberine treatment reduced the apoptotic rate in LBer group, MBer group and HBer group in a concentration-dependent manner. Compared with Con group, the protein levels of PTEN and cleaved caspase-3 increased, while PI3K and phosphorylation of Akt decreased significantly in Axn group. However, this effect was reversed by berberine in a concentration-dependent manner. Compared with Con group, the activation of HNF-1α/PDX-1 signaling pathway was inhibited in Axn group but recovered by berberine administration. The abilities of releasing insulin under normal or high-glucose stimulation were impaired in Axn group but recovered by berberine treatment in LBer group, MBer group and HBer group in a concentration-dependent manner.CONCLUSION: Berberine shows protective effects against alloxan-induced damage in beta cells by inhibiting apoptosis and recovering insulin secretion, thus attenuating the activation of PTEN/PI3K/Akt and HNF-1α/PDX-1 signaling pathways.     

丰产早熟四倍体长豇豆新品种一桶天下的选育

采用系谱法对秋水仙素化学诱变的米美小叶进行选育,获得四倍体长豇豆一桶天下。植株蔓生,生长势强,平均株高306.5 cm,节间长度20.2 cm。早熟,始花节位位于第3节,生育期93 d(天)左右,出苗至始收58 d(天)左右。连续结荚能力强,双荚四荚率高,商品荚绿白色,荚面光滑有光泽,缝线不显,喙红色,无鼠尾,不鼓籽,荚长89.3 cm左右,荚粗0.9cm左右,平均单荚质量27.4 g,单株结荚20根左右,每667 m~2产量2 200 kg左右。田间对病毒病、白粉病和锈病的抗性强于对照早豇4号,综合性状表现优良,适宜在江苏、浙江、安徽、河南等地栽培。     

Inhibitory effect of madecassoside on LPS-stimulated microglia

AIM: To observe the inhibitory effect of madecassoside on the LPS-stimulated microglia and to investigate its possible mechanism. METHODS: Microglia cells of neonatal Sprague-Dawley (SD) rats were cultured, isolated and purified. Microglia cells were activated with lipopolysaccharide (LPS). The inhibitory effect of madecassoside on microglia was measured by MTT assay. Tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β) were detected by ELISA. Cell cycle and apoptotic rate were evaluated by flow cytometry. The expression of TLR4 was detected by Western blotting. The expression of NF-κB was detected by RT-PCR. RESULTS: LPS induced the proliferation of microglia and release inflammatory cytokines significantly. Compared with LPS group, madecassoside inhibited the proliferation of microglia induced by LPS in a dose dependent manner. The IC₅₀ value of madecassoside was 10.97 nmol/L to microglia after incubation for 48 h. Madecassoside also decreased the levels of TNF-α and IL-6, increased the ratios of microglia at the G₂ phase and the apoptotic rate, decreased the expression of TLR4 and NF-κB significantly (P<0.05). CONCLUSION: Madecassoside has inhibitory effects on the proliferation of LPS-stimulated microglia, by which the mechanism may be related to inhibition of the expression of TLR4 and NF-κB, change of cell cycle distribution and induction of microglia apoptosis.     

Therapeutic effect of MAG gene silencing by siRNA on remyelination and motor recovery after intracerebral hemorrhage in rats

AIM:To study the functional rehabilitative effects of lentiviral vector-mediated siRNA silencing myelin-associated glycoprotein (MAG) on neurological improvement and remyelination after intracerebral hemorrhage (ICH) in rats. METHODS:ICH was induced in 45 adult male Sprague-Dawley rats by a stereotactically guided injection of bacterial type IV collagenase into the right internal capsule. Lentiviral vector-mediated siRNA targeting MAG (siRNA), or PBS was administered left intracerebroventricularly 1 d after right internal capsule ICH. The Rotarod motor test scores were determined 1 d before and 1 d, 7 d, 14 d and 21 d after treatment. Luxol fast blue staining, immunohistological staining and Western blotting were used to detect the changes of MAG. RESULTS:Compared with PBS group, the changes of Rotarod motor test scores indicated that the ICH rats receiving siRNA had significant functional recoveries. As indicated by Luxol fast blue staining, the area of demyelination in percentage was obviously reduced in siRNA treatment group as compared with PBS group after ICH (P<0.05). The expression of MAG detected by immunohistological staining and Western blotting was significantly lower in siRNA-treated brain than that in PBS group (P<0.05). CONCLUSION:siRNA targeting MAG contributes to the remarkable functional recovery after ICH. The treatment promotes nerve fiber remyelination by down-regulation of the MAG expression after ICH in rats.     

矮牵牛花发育相关基因PhTs2的克隆及功能分析

从矮牵牛‘幻想’中克隆了花发育相关基因PhTs2，该基因CDS长855 bp，编码284个氨基酸，具有保守的adh-short结构域。进化分析表明，PhTs2蛋白与同科植物烟草和番茄的同源蛋白亲缘关系较近。实时荧光定量PCR分析发现该基因在矮牵牛花药发育的早期特异性表达。为分析其功能，构建PhTs2超量表达载体，转化了烟草，并对转基因烟草进行了鉴定：表型观测发现其花药形态异常，花粉数量减少；花粉活力和萌发率极显著降低；扫描电镜观测发现其花粉粒畸形，外壁纹饰不规则；半薄切片显示其绒毡层发育过程紊乱。该研究为培育雄性不育系提供了潜在的基因资源。     

Inhibitory effects of dexamethasone on the expression of integrin CD18 induced by PMA

AIM:To study the effect of dexamethasone(Dex) on the expression of CD18. METHODS:Quantitative RT-PCR analysis, Northern blotting technique were used to measure the expression of CD18 in U937 cells treated by PMA.RESULTS:Dex could significantly attenuated the effects of PMA in a dose-dependent manner (10^-6 mol/L-10^-10 mol/L). These effects of Dex (10^-7 mol/L) were completely aborted by RU-486 (10^-6 mol/L).CONCLUSION:Dex, via GR, could inhibit CD18 mRNA expression in U937 cells treated by PMA. The effects of Dex might be possibly depended on the counteracting action on the NF-κB.     

Mechanism of systemic inflammatory response syndrome induced by EC DNA

AIM: To investigate whether the bacterial DNA participates in SIRS and its possible mechanism. METHODS: Escherichia coli genomic DNA (EC DNA) was extracted and purified from Escherichia coli 25922 by alkaline lysis method. Mortality of mice challenged with EC DNA and the changes of TNF-α and IL-6 in rat serum were observed. ANA-1 cells were cultured in vitro, after the cells were stimulated by different concentrations of EC DNA and LPS, the level of TNF-α and IL-6 in supernatant were tested. Meanwhile,expression of TLR9 and TLR4 on cell surface was measured. Activation of NF-JB was also observed. RESULTS: The lethal effect of EC DNA on mice with an obvious dose-effect relationship was observed. The death happened within 24 hours. Calf thymus DNA and DNase I-treated EC DNA did not lead to mice to die. The changes of serum TNF-α and IL-6 in rats induced by EC DNA and LPS were similar, but TNF-α peak level of EC DNA group appeared 1 hour earlier than that of LPS group. In vitro, large amount of TNF-α and IL-6 were released from ANA-1 cells stimulated by EC DNA. High expression of TLR9 and TLR4 was observed on surfaces of THP-1 cells. In particularly, LPS induced strong activation of NFκB. The results suggested other pathway possibly took part in the signal transduction inducea by EC DNA. CONCLUSION: EC DNA has the abilities to lead to death of mice, andinduces serum TNF- αand IL- 6 level to increase in rats and ANA- 1 cells to release cytokines in vitro. High expression of TLR9 and TLR4, strong activation of NF- κB may be its importantmolecular mechanism, but other pathway probably exists to play an important role.     

Effects of tetrahydroxystilbene-2-O-β-D-glucoside on apoptosis and expression of bcl-2/bax/caspase-3 in HUVECs treated with homocysteine

AIM：To explore the effects of tetrahydroxystilbene-2-O-β-D-glucoside (TSG) from Polygonum multiflorum on the apoptosis and the mRNA expression of bcl-2, bax and caspase-3 in human umbilical vein endothelial cells (HUVECs) treated with homocysteine (Hcy). METHODS：Cultured HUVECs were treated with Hcy (3 mmol/L) to establish a Hcy-damaged model. HUVECs in TSG treated groups were pre-incubated with TSG at concentrations of 1 μmol/L and 10 μmol/L for 2 h before treated with Hcy. Cell nuclear damage was detected by Hoechst 33342 staining. Cell apoptosis was determined by flow cytometry. The mRNA expression of bcl-2, bax and caspase-3 was measured by real-time fluorescence quantitative RT-PCR. RESULTS： After treatment with Hcy at concentration of 3 mmol/L, the nuclear damage and apoptotic rate of HUVECs were higher than that in normal group. The expression of bcl-2 was lower, and the expression of Bax and caspase-3 was higher than that in normal group. On the other hand, pre-incubation with TSG at concentrations of 1 μmol/L and 10 μmol/L decreased the nuclear damage and cell apoptosis, increased the expression of bcl-2, and decreased the expression of bax and caspase-3 as compared with the cells only treated with Hcy. CONCLUSION：TSG reduces the apoptosis of HUVECs induced by Hcy, and the mechanism might be associated with regulating the expression of bcl-2, bax and caspase-3.     

Effects of bortezomib on Treg cells in multiple myeloma and its relationship with tumor burden and chemoresponse

AIM: To investigate the changes of regulatory T (Treg) cells in peripheral blood from multiple myeloma (MM) patients with bortezomib-based treatment, and to analyze the effects of bortezomib on Treg cells in MM and the relationship with tumor burden and chemoresponse. METHODS: Treg cells from 30 newly-diagnosed MM patients were detected by flow cytometry before and after bortezomib-based treatment. Meanwhile, 12 cases with conventional treatment were enrolled as controls. The correlation of Treg cell count with the levels of M protein and serum β₂-microglobulin (β₂-MG) was then statistically analyzed. RESULTS: The patients in early disease stage had lower Treg cell count. Decreased Treg cell count was found in 31 cases with chemoresponse (P<0.01), which did not exist in the 11 cases without chemoresponse. The patients achieved >50% serum β₂-MG reduction showed more obvious changes of Treg cell count than those achieved ≤50% serum β₂-MG reduction (P<0.05). The patients with bortezomib-based treatment showed more apparent decline in Treg cell count than the patients with conventional treatment, which were related to chemoresponse and serum β₂-MG reduction (P<0.05). CONCLUSION: Treg cell count is a reliable index of tumor budern. Both bortezomib-related treatment and conventional treatment can reduce Treg cell count, but the reduction is more obvious in bortezomib-related treatment. Such reduction is related to chemoresponse and tumor burden, suggesting the mechanism of bortezomib curing MM may be by the way of Treg suppression.     

Effect of cimicifugoside H-1 on amino acid neurotransmitters in striatum of rats with cerebral ischemia

AIM: To study the neuroprotective effect of cimicifugoside H-1 and to explore the mechanism involved by determining the variation of amino acid neurotransmitters in extracellular fluid in the striatum of rats with cerebral ischemia. METHODS: The rats were randomly divided into sham-operated, cerebral ischemia, high-, middle- and low-dose cimicifugoside H-1, and ginkgo groups. Focal cerebral ischemia model was established by middle cerebral artery occlusion (MCAO) with sutures. Normal saline was intraperitoneally injected into the rats in sham-operated group and cerebral ischemia group, while ginkgo and different doses of cimicifugoside H-1 were injected into the rats in ginkgo group and high-, middle- and low-dose cimicifugoside H-1 groups, respectively, once a day for 7 d. The striatal fluids were gained in vivo by brain microdialysis. The contents of aspartic acid, glutamic acid, glycine and γ-aminobutyric acid were tested by high-performance liquid chromatography electrochemical detector system. RESULTS: Compared with sham-operated group, the contents of excitatory amino acids (aspartic acid and glutamic acid) were significantly increased 2 h after cerebral ischemia (P<0.05). Compared with cerebral ischemia group, the contents of aspartic acid and glutamic acid were significantly decreased 2 h after cerebral ischemia in high-dose cimicifugoside H-1 and ginkgo groups (P<0.05). Compared with cerebral ischemia group, the contents of aspartic acid and glutamic acid did not show significant decrease 2 h after cerebral ischemia in middle- and low-dose cimicifugoside H-1 groups. Compared with sham-operated group, the contents of inhibitory amino acid (γ-aminobutyric acid and glycine) were significantly decreased 3 h after cerebral ischemia in cerebral ischemia group (P<0.05). Compared with cerebral ischemia group, the contents of γ-aminobutyric acid and glycine were significantly increased 3 h after cerebral ischemia in high-dose cimicifugoside H-1 and ginkgo groups (P<0.05). Compared with cerebral ischemia group, the contents of γ-aminobutyric acid and glycine did not show significant decrease 3 h after cerebral ischemia in middle- and low-dose cimicifugoside H-1 groups. CONCLUSION: Cimicifugoside H-1 restrains the excessive releases of excitatory amino acids and increases the contents of inhibitory amino acids during cerebral ischemia. It doesn't only penetrate through the blood brain barrier, but also regulates the disorder of excitatory amino acid during cerebral ischemia, thus showing the protective function to cerebral neuron during cerebral ischemia.     

黄瓜籽化学成分及其功效

黄瓜籽中富含脂肪酸、植物甾醇、糖和苷类、挥发油类、无机元素等营养成分，是一种营养保健食品。此外，黄瓜籽还具有极高的药用价值。本文综述了黄瓜籽化学成分及其各种功效的研究进展和所取得的成果，并对其未来的发展方向进行了展望。     

Inhibitory effect of proanthocyanidins B1 and B2 on inflammation of BV-2 cells induced by lipopolysaccharide

AIM: To investigate the different inhibitory effects of proanthocyanidins B1 and B2, which are isomers, on the inflammatory response of BV-2 cells induced by lipopolysaccharide (LPS). METHODS: MTT assay was used to detect the effects of proanthocyanidins B1 and B2 on the viability of BV-2 cells. LPS (1 mg/L) was used to promote BV-2 cells to secrete inflammatory factors. ELISA, chemotaxis assay and Western blot were used to detect the influence of proanthocyanidins B1 and B2 on the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cell chemotaxis and phosphorylation of NF-κB. RESULTS: Proanthocyanidins B1 and B2 did not show cytotoxicity effect on BV-2 cells. Proanthocyanidin B1 and B2 inhibited the cell chemotaxis, phosphorylation of NF-κB, and releases of TNF-α and IL-1β. CONCLUSION: Proanthocyanidins B1 and B2 inhibit the inflammatory response of BV-2 cells induced by LPS, and their action intensity didn't show significant difference.     

Effects of hydrogen sulfide on impaired wound healing in ob/ob mice

AIM: To investigate the role of hydrogen sulfide(H₂S) on impaired wound healing in ob/ob mice and the underlying mechanism.METHODS: The ob/ob mice were randomly divided into 3 groups, including vehicle, insulin and NaHS for treatment. C57BL/6 mice were treated with vehicle as control. Full-thickness punch biopsy wounds were created on the mice. Firstly, H₂S concentrations in the skins and granulation tissues were measured. The mRNA expression of cystathionine γ-lyase(CSE) was detected by RT-qPCR. The protein expression of CSE and MMP-9 were determined by Western blot. The neutrophil and monocyte/macrophage infiltration was analyzed by immunohistochemistry me-thod. The levels of tumor necrosis factor(TNF)-α and interleukin(IL)-6 were measured by ELISA.Collagen formation was measured by Masson staining.RESULTS: The H₂S levels in the skin and granulation were significantly decreased in ob/ob mice and increased in the NaHS-treated mice(P<0.05). CSE expression at mRNA and protein levels was significantly decreased in ob/ob mice compared with the control mice(P<0.05). The wound healing period was significantly shorter in NaHS group than that in vehicle-treated ob/ob mice group(P<0.05), in which the insulin group had no difference with vehicle ob/ob mice group. The neutrophil and monocyte/macrophage infiltration, and TNF-α and IL-6 levels were significantly increased in ob/ob groups, but were decreased in NaHS group(P<0.01 or P<0.05). Meanwhile, NaHS increased collagen formation in the granulation tissues of ob/ob mice.CONCLUSION: H₂S/CSE down-regulation contributes to impaired wound healing in diabetes, which is alleviated by exogenous H₂S possibly through anti-inflammation.