虫草素对肺癌细胞生长及迁移的影响

探讨虫草素对非小细胞肺癌细胞株H1781细胞凋亡及迁移的影响及作用机制。培养H1781细胞并分组,对照组用不含药物的培养基处理,虫草素组用含有10、20、30和40 μmol/L虫草素处理,处理24 h后测定细胞活力,通过显微镜观察细胞形态学,HE染色观察虫草素对细胞整体的影响,细胞免疫荧光技术检测细胞中MMP-9和DAPI核染色情况观察细胞凋亡,Western blotting检测凋亡等相关蛋白表达。与对照组相比,虫草素处理24 h后,H1781细胞系活力显著降低;细胞数量明显减少;HE染色观察发现随着虫草素浓度增加,细胞数量及细胞集团明显变少,免疫荧光技术检测发现药物处理后细胞凋亡明显促进;划痕实验发现虫草素明显降低细胞迁移能力;Western blotting实验中Bax、cleaved caspase-3蛋白表达明显上调,MMP-9、Bcl-2蛋白表达明显下调。虫草素对肺癌H1781细胞迁移有抑制作用、对凋亡有促进作用,推测其作用机制为上调促凋亡蛋白表达、下调抗凋亡蛋白表达。     

稳定表达人源GABAAR-CHO细胞株的建立

目的: 构建α₁亚基诱导表达、β₂和γ_2L亚基稳定表达的人源α₁β₂γ_2L-GABA_AR-CHO(Chinese hamster ovary)细胞株。方法: 从人cDNA文库中扩增α₁、β₂、γ_2L亚基编码基因,分别构建亚基表达载体;将三个亚基表达载体共转染CHO-K1细胞,通过抗性筛选、膜电位检测法进行稳定表达克隆筛选;通过qPCR、Western blot对亚基表达进行鉴定;以激动剂GABA、阳性变构调节剂地西泮(diazepam,Dia)、拮抗剂荷包牡丹碱(bicuculine)为工具药,采用全细胞膜片钳方法及膜电位检测法对稳定表达细胞的药理学功能进行鉴定。结果: 经克隆筛选获得表达量较高的α₁β₂γ_2L-GABA_AR-CHO并对其亚基表达鉴定,结果显示该细胞稳定表达α₁、β₂、γ_2L亚基,构建的α₁β₂γ_2L-GABA_AR-CHO细胞仅在加入四环素(tetracyclin)诱导的情况下表达α₁亚基并与β₂、γ_2L组装成具有功能活性的α₁β₂γ_2L-GABA_AR;对其进行全细胞膜片钳检测研究发现,GABA可对其产生激动效应,引起α₁β₂γ_2L-GABA_AR-CHO细胞产生氯离子通道特征性电流变化,Dia可剂量依赖性地增强GABA对α₁β₂γ_2L-GABA_AR的激动效应;在膜电位检测研究中,获得GABA激动效应EC₅₀为(177.72 ± 15.92)nmol/L,Dia变构效应EC₅₀为(3.63±0.52)μmol/L,拮抗剂Bicuculine拮抗效应IC₅₀为(538.83±29.55)nmol/L。结论: 通过采用诱导表达策略,成功构建了α₁β₂γ_2L-GABA_AR-CHO稳定表达细胞株,该细胞株具有对激动剂、阳性变构剂、拮抗剂特异性检测的药理学功能。     

奶油栓孔菌菌丝体多糖的提取工艺优化、结构表征及抗氧化活性

奶油栓孔菌Trametes lactinea是一种生物活性丰富的大型真菌。本研究在单因素试验的基础上,通过响应面法优化其菌丝体多糖的提取工艺,利用DEAE-Cellulose-52阴离子交换柱和Sephadex G-200层析柱对粗多糖进行分离纯化,获得TLMPS-0、TLMPS-1和TLMPS-3均一多糖组分。采用化学组成分析、UV-vis、FTIR、刚果红实验对3种多糖组分进行结构分析,并检测了多糖清除自由基的能力和还原力。结果表明,奶油栓孔菌菌丝体多糖最优提取工艺为：提取温度99℃、料液比1:30 (g/mL)、提取时间5h,提取次数2次。在此工艺条件下,多糖提取率为4.01%。TLMPS-0、TLMPS-1和TLMPS-3的糖醛酸含量分别为12.91%±0.44%、8.24%±0.22%、7.50%±0.66%,硫酸基含量分别为22.24%±1.88%、14.55%±0.56%、18.68%±0.69%,并且证明TLMPS-0是一种α-吡喃型多糖或β-吡喃型多糖,而TLMPS-1是一种β-吡喃型多糖,均不具备三螺旋空间构象,此外,3种多糖组分均具有一定的清除DPPH自由基、ABTS自由基、羟基自由基的能力和铁离子还原能力,其中TLMPS-0抗氧化活性最强。研究结果为奶油栓孔菌多糖的功能研究与挖掘提供了研究基础与理论依据。     

TOFA对人食管鳞癌Eca109和KYSE-450细胞生长的影响*

目的: 探讨5-十四烷氧基-2-呋喃酸(TOFA)对人食管鳞癌(ESCC)细胞Eca109和KYSE-450细胞增殖、周期和凋亡的影响。方法: 将Eca-109细胞和KYSE-450细胞分为对照组(DMSO)和实验组(TOFA),细胞(4×10³ cells/100 μl)接种于96孔板中,每个浓度设置5个复孔,培养24 h后,给予DMSO(对照)和不同浓度(1、3、5、10 μg/ml)TOFA处理,继续培养24、48和72 h;MTT检测细胞增殖,流式细胞术检测细胞周期和凋亡,Western blot检测p21、Cleaved caspase-3表达水平及p-AKT、p-mTOR、p-4EBP1修饰水平,专用试剂盒检测细胞内游离脂肪酸。结果: 与DMSO组比较,TOFA以浓度和时间依赖性方式抑制Eca109和KYSE-450细胞增殖(P均<0.05),处理48 h的IC₅₀分别为4.65和3.93 μg / ml;实验组细胞G2 / M 期细胞百分比增加,细胞凋亡率增高,p21、Cleaved caspase-3蛋白表达水平上调(P均<0.05),p-AKT、p-mTOR、p-4EBP1修饰水平下调(P均<0.05)。结论: TOFA抑制人食管鳞癌细胞增殖、阻滞细胞周期并促进细胞凋亡,其机制可能与其抑制AKT/mTOR/4EBP1信号通路有关。     

红托竹荪多糖诱导肿瘤细胞凋亡的作用初探

探讨红托竹荪多糖对小鼠腹水瘤S180细胞凋亡的作用。分别使用不同浓度的红托竹荪多糖处理S180细胞24h,Western blot法检测Bcl-xl、Bax、caspase-9和Caspase-3等蛋白表达,使用流式细胞仪检测细胞凋亡。Western blot 法检测结果显示Bcl-xl表达量随竹荪多糖剂量增加而降低,Bax表达量则相反,每组Bax/Bcl-xl的比值增加,Caspase-9、Caspase-3表达量增加;流式细胞仪检测结果显示：0、25、50和100mg/L组细胞凋亡率分别为5.12%±0.11%、9.61%±0.61%、16.39%±0.19%和17.05%±0.13%,与对照组相比细胞凋亡率增高,差异具有统计学意义（P<0.05）,推断红托竹荪多糖具有诱导S180细胞凋亡的功能。     

紫陀螺菌化学成分及其抑制肿瘤细胞增殖活性的研究

以紫陀螺菌为对象,研究其子实体的化学成分及其抑制肿瘤细胞增殖活性。采用溶剂提取、柱层析和高效液相色谱等方法分离纯化化学成分,通过核磁共振和质谱技术鉴定单体化合物结构,运用结晶紫法评价单体化合物抑制肿瘤细胞增殖活性。从乙酸乙酯提取物中共分离鉴定6个单体化合物,分别为(22E,24R)-麦角甾-5,7,22-三烯-3β-醇(1)、3β,5α-二羟基-(22E,24R)-麦角甾-7,22-二烯-6-酮(2)、(22E,24R)-麦角甾-7,22-二烯-3β,5α,6β-三醇(3)、吲哚-3-甲酸甲酯(4)、4,4-二甲基-1,7-庚二酸(5)和(8E,10E)-12羰基十八碳-8,10-二烯酸(6),其中化合物1为主要成分,相对含量为23.8%。活性测试结果表明3对人乳腺癌细胞株MCF-7 细胞、人胰腺癌细胞株PANC-1细胞和人乳腺癌细胞株MDA-MB-231细胞具有微弱的细胞增殖抑制活性。本研究首次报道了紫陀螺菌化学成分,对深入挖掘其在健康领域中的开发价值具有重要意义。     

7株野生虫草菌的鉴定及其菌丝体醇提取物对HepG2细胞的抑制活性

结合形态学与ITS序列分析对7株野生虫草真菌进行分类鉴定。MTT法分析它们的菌丝体醇提取物对肝癌HepG2细胞增殖的抑制活性。鉴定结果表明菌株MF7、MF9、MF14为细脚棒束孢Isaria tenuipes,菌株MF11、MF12、MF13为蝉棒束孢Isaria cicadae,菌株MF10为球孢白僵菌Beauveria bassiana;MTT结果显示分离到的3株细脚棒束孢和3株蝉棒束孢的菌丝体醇提取物对HepG2的抑制活性较差,IC₅₀均大于500μg/mL;球孢白僵菌MF10对HepG2细胞有一定抑制作用,IC₅₀值为221.6μg/mL,略强于蝙蝠蛾拟青霉发酵菌丝粉产品金水宝胶囊（IC₅₀=364μg/mL）和中华被毛孢发酵菌丝粉产品百令胶囊（IC₅₀=268.7μg/mL）。另外,发现供对比试验的3株蛹虫草菌株（MF1、MF5、MF15）对HepG2细胞均有较好的抑制作用,其中MF15的发酵菌丝体醇提取物活性最强,IC₅₀为55.56μg/mL,暗示蛹虫草发酵菌丝体具有重要的研究价值。     

RS4651通过上调SMAD7抑制小鼠肝细胞AML12的EMT作用

目的: 研究RS4651对小鼠肝细胞AML12的EMT、细胞增殖以及迁移能力的影响和作用机制。方法: 以不同浓度的RS4651干预小鼠AML12细胞,通过Western blot和RT-PCR法检测RS4651各浓度组及空白对照组细胞EMT指标E-cadherin、N-cadherin、Vimentin的表达水平。RNA-sequencing确定RS4651的作用通路及互作网络的关键性节点基因。应用SMAD7-siRNA构建SMAD7敲低的AML12细胞模型,细胞分为对照组、RS(60 μmol/L)组、SMAD7-siRNA组以及SMAD7-siRNA+RS(60 μmol/L)组,应用Western blot和RT-PCR法分别检测各组细胞EMT指标。进一步应用细胞增殖MTS法和Transwell小室迁移法检测各组细胞的增殖和迁移能力,比较各组间差异。结果: RS4651呈浓度依赖性抑制AML12细胞EMT,TGF-β1信号通路中的SMAD7是RS4651发挥作用的关键性节点。敲低SMAD7后,RS4651对AML12细胞EMT、增殖和迁移的抑制作用减弱。结论: RS4651可以通过上调SMAD7抑制小鼠肝细胞EMT、增殖和迁移。     

二甲双胍联合阿霉素应用对人乳腺癌细胞增殖和凋亡的影响

目的：观察二甲双胍联合阿霉素应用对人乳腺癌细胞MDA-MB-231增殖和凋亡的影响。方法：MTT法分别检测二甲双胍、阿霉素和二甲双胍联合阿霉素对MDA-MB-231细胞生长的抑制作用;平板克隆实验检测二甲双胍联合阿霉素对MDA-MB-231细胞克隆形成能力的影响;流式细胞仪检测二甲双胍联合阿霉素对MDA-MB-231细胞凋亡的影响。结果：二甲双胍和阿霉素分别对MDA-MB-231细胞生长有抑制作用,二甲双胍联合阿霉素应用对MDA-MB-231细胞生长的抑制作用更加显著,并且随着药物浓度的增加而增加;二甲双胍联合阿霉素应用与单药相比能够明显降低MDA-MB-231细胞克隆形成率,并且促进细胞凋亡。结论：二甲双胍联合阿霉素应用与单药相比能够显著抑制人乳腺癌细胞MDA-MB-231细胞的增值,促进其凋亡,可见两药联用对肿瘤细胞的杀伤具有协同性。     

大鼠SUMO特异性蛋白酶1催化区蛋白制备及功能鉴定*

目的: 制备大鼠SUMO特异性蛋白酶1(sentrin-specific protease,SENP1)催化结构域(SENP1C)蛋白,并鉴定其酶活性。方法: 分别以大鼠SENP1-pcDNA3.1和EGFP-pcDNA3.1重组体为模板,PCR扩增目的基因,克隆入pGEM-T载体;酶切鉴定后,再亚克隆入原核表达载体pET-28a;阳性重组体导入原核表达细胞BL-21,异丙基硫半乳糖苷(IPTG)诱导蛋白质表达;SDS-PAGE及考马斯亮蓝染色鉴定蛋白质的表达。Ni-NTA吸附纯化蛋白质并透析处理,SDS-PAGE及考马斯亮蓝染色鉴定蛋白质的纯度;1 μmol/L及5 μmol/L Tat-EGFP分别孵育HT22细胞不同时间,荧光显微镜下观察细胞转染情况。采用5 μmol/L Tat-SENP1C预孵育HT22细胞10 h,免疫印迹检测整体蛋白质的SUMO化水平;用5 μmol/L Tat-SENP1C预孵育HT22细胞或过表达Myc-Akt1和HA-SUMO1的HT22细胞10 h后,免疫沉淀和免疫印迹检测内源性和外源性Akt1与SUMO1的结合(SUMO化)。结果: Tat-SENP1C-pET-28a和Tat-EGFP-pET-28a重组原核表达载体成功构建,IPTG可以诱导蛋白质高表达;采用Ni-NTA纯化和透析可获得较高纯度的蛋白质;5 μmol/L Tat-EGFP孵育HT22 细胞10 h后,蛋白质穿膜效率较高;Tat-SENP1C重组蛋白可以显著降低HT22细胞中整体蛋白质的SUMO化以及内源性和外源性Akt1 SUMO化。结论: Tat-SENP1C-pET-28a和Tat-EGFP-pET-28a重组原核表达载体构建成功,且被IPTG诱导后可高效表达蛋白质;纯化的Tat-SENP1C蛋白具有较强的穿膜能力及酶活性。     

Protocol for the measurement of fatty acid and glycerol turnover in vivo in baboons

Recognition of the strength of nonhuman primate models in investigating metabolic disorders has resulted in an expanded need for in vivo research techniques. We studied adipose metabolism in 10 baboons (13.0 ± 4.2 years old, 29.5 ± 5.5 kg). Part 1 evaluated the effect of different sedatives on the rate of appearance of plasma free fatty acids (RaFFA), assessed using ¹³C₄-labeled palmitate infusion (7 µmol/kg/min). Animals, were studied with no sedation, with complete isoflurane sedation, and with minimal midazolam infusion (0.04 mg/kg/h), with the last scheme allowing for the most consistent values and animals that were visually more calm. In Part 2, RaFFA and RaGlycerol (D₅-glycerol, 5 mg/kg lean body mass/h) were measured. From midnight to 0300, flux fell and came to a steady state between 0500 and 0700 h (RaFFA, 39.4 ± 29.8 μmol/kg fat mass/min; and RaGlycerol, 26.9 ± 7.3 μmol/kg/min). The RaFFA-to-RaGlycerol ratio was 1.5 ± 0.8 (49% reesterification). The decline in turnover throughout the night reflects natural circadian processes and was mirrored by reductions in FFA and glycerol to 0.62 and ± 0.14 and 0.16 and ± 0.03 mmol/l, respectively. The concurrent changes in both FFA and glycerol kinetics indicate physiologic validity of the method. These techniques will support needed research to determine mechanisms by which treatments act upon the adipocyte in vivo.     

Lipo-Endomorphin-1 Derivatives with Systemic Activity against Neuropathic Pain without Producing Constipation

To enhance the drug-like properties of the endogenous opioid peptide endomorphin-1 (1 = Tyr-Pro-Trp-Phe-NH₂), the N-terminus of the peptide was modified with 2-aminodecanoic acid, resulting in compound 3. Tyr in compound 1 was replaced with 2,6-dimethyltyrosine yielding compound 2. Derivative 2 was also substituted with 2-aminodecanoic acid producing compound, 4. Lipoamino acid-modified derivatives showed improved metabolic stability and membrane permeability while maintaining high μ-opioid (MOP) receptor binding affinity and acting as a potent agonist. In vivo studies showed dose-dependent antinociceptive activity following intravenous (i.v.) administration of compounds 3 and 4 in a chronic constriction injury (CCI)-rat model of neuropathic pain with ED₅₀ values of 1.22 (±0.93) and 0.99 (±0.89) µmol/kg, respectively. Pre-treatment of animals with naloxone hydrochloride significantly attenuated the anti-neuropathic effects of compound 3, confirming the key role of opioid receptors in mediating antinociception. In contrast to morphine, no significant constipation was produced following i.v. administration of compound 3 at 16 µmol/kg. Furthermore, following chronic administration of equi-potent doses of compound 3 and morphine to rats, there was less antinociceptive tolerance for compound 3 compared with morphine.     

Overexpression of SDF-1�� Enhanced Migration and Engraftment of Cardiac Stem Cells and Reduced Infarcted Size via CXCR4/PI3K Pathway

Cardiac stem cells (CSCs) can home to the infarcted area and regenerate myocardium. Stromal cell-derived factor-1α/C-X-C chemokine receptor type 4 (SDF-1α/CXCR4) axis is pivotal in inducing CSCs migration. However, the mechanisms remain unclear. This study set out to detect if SDF-1α promotes migration and engraftment of CSCs through the CXCR4/PI3K (phosphatidylinositol 3-kinase) pathway. In the in vitro experiment, c-kit+ cells were isolated from neonatal mouse heart fragment culture by magnetic cell sorting. Fluorescence-activated cell sorting results demonstrated that a few c-kit+ cells expressed CD45 (4.54%) and Sca-1 (2.58%), the hematopoietic stem cell marker. Conditioned culture could induce c-kit+ cells multipotent differentiation, which was confirmed by cardiac troponin I (cTn-I), α-smooth muscle actin (α-SMA), and von Willebrand factor (vWF) staining. In vitro chemotaxis assays were performed using Transwell cell chambers to detect CSCs migration. The results showed that the cardiomyocytes infected with rAAV1-SDF-1α-eGFP significantly increased SDF-1α concentration, 5-fold more in supernatant than that in the control group, and subsequently attracted more CSCs migration. This effect was diminished by administration of AMD3100 (10 µg/ml, CXCR4 antagonist) or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (20 µmol/L, PI3K inhibitor). In myocardial infarction mice, overexpression of SDF-1α in the infarcted area by rAAV1-SDF-1α-eGFP infection resulted in more CSCs retention to the infarcted myocardium, a higher percentage of proliferation, and reduced infarcted area which was attenuated by AMD3100 or ly294002 pretreatment. These results indicated that overexpression of SDF-1α enhanced CSCs migration in vitro and engraftment of transplanted CSCs and reduced infarcted size via CXCR4/PI3K pathway.     

Adsorption and Degradation of Doxorubicin from Aqueous Solution in Polypropylene Containers

The purpose of this study was to examine doxorubicin adsorption in polypropylene containers as a function of pH and drug concentration based on anecdotal evidence of such adsorption. Doxorubicin loss was first examined in high-performance liquid chromatography (HPLC) glass inserts by UV absorbance to determine appropriate pH and time durations for subsequent analysis. Doxorubicin loss was then investigated in polypropylene microcentrifuge tubes at different pH values and starting drug concentration at 37°C over 48 h using HPLC with fluorescent detection. Doxorubicin concentrations was essentially constant in HPLC glass inserts at pH 4.8 up to 12 h but declined 5% at pH 7.4 by 3 h. The percent doxorubicin adsorption was calculated in polypropylene microcentrifuge tubes from extrapolations to zero time and was the least at pH 4.8, but increased with pH values 6.5 and 7.4, and decreased with drug concentration to reach a maximum adsorption of 45% in 2.0 μg/mL at pH 7.4 and 37°C. Degradation rate constants, ranging from 0.0021 to 0.019 h⁻¹, also increased with pH in these studies. Determinations of low amounts of doxorubicin in polypropylene containers at pH 7.4 may be underestimated if adsorption and degradation issues are not taken into account.KEY WORDS: adsorption, analysis, chemical stability, doxorubicin, glass, HPLC, polypropylene     

北亚热带地带性森林土壤温室气体通量对土地利用方式改变和降水减少的响应

弄清土地利用和降水变化对林地土壤主要温室气体(CO₂、CH₄和N₂O)排放通量变化的影响, 是准确评估森林土壤温室气体排放能力的重要基础。该研究以常绿落叶阔叶混交林原始林、桦木(Betula luminifera)次生林和马尾松(Pinus massoniana)人工林为对象, 采用静态箱-气相色谱法研究了3种土地利用方式(常绿落叶阔叶混交林原始林、桦木次生林和马尾松人工林)和降水减少处理状况下森林土壤CO₂、CH₄和N₂O通量排放特征, 并探讨了其环境驱动机制。研究结果表明: 原始林土壤CH₄吸收通量显著高于次生林和人工林, 次生林CH₄吸收通量显著高于人工林土壤。人工林土壤CO₂排放通量显著高于原始林和次生林土壤。次生林土壤N₂O排放通量高于原始林和人工林, 但三者间差异不显著。降水减半显著抑制了3种不同土地利用方式下林地土壤CH₄吸收通量; 降水减半处理对原始林和次生林土壤CO₂排放通量均具有显著的促进作用, 而对人工林土壤CO₂排放通量具有显著的抑制作用; 降水减半处理促进了原始林和人工林林地土壤N₂O排放而抑制了次生林林地土壤N₂O排放。原始林和次生林林地土壤CH₄吸收通量随土壤温度升高显著增加, CH₄吸收通量与土壤温度均呈显著相关关系; 原始林、次生林和人工林土壤CO₂和N₂O排放通量与土壤温度均呈显著正相关关系; 土壤湿度抑制了次生林和人工林土壤CH₄吸收通量, 其CH₄吸收通量随土壤湿度增加显著减少; 原始林土壤CO₂排放通量与土壤湿度呈显著正相关关系。自然状态下, 原始林土壤N₂O排放通量与土壤湿度呈显著正相关关系, 原始林和次生林土壤N₂O排放通量与硝态氮含量呈显著相关关系。研究结果表明全球气候变化(如降水变化)和土地利用方式的转变将对北亚热带森林林地土壤温室气体排放通量产生显著的影响。     

Greenhouse gas fluxes of typical northern subtropical forest soils: Impacts of land use change and reduced precipitation

Aims It is important to study the effects of land use change and reduced precipitation on greenhouse gas fluxes (CO₂, CH₄ and N₂O) of forest soils. Methods The fluxes of CO₂, CH₄ and N₂O and their responses to environmental factors of primary forest soil, secondary forest soil and artificial forest soil under a reduced precipitation regime were explored using the static chamber and gas chromatography methods during the period from January to December in 2014. Important findings Results indicate that CH₄ uptake of primary forest soil ((-44.43 ± 8.73) μg C·m^-2·h^-1) was significantly higher than that of the secondary forest soil ((-21.64 ± 4.86) μg C·m^-2·h^-1) and the artificial forest soil ((-10.52 ± 2.11) μg C·m^-2·h^-1). CH₄ uptake of the secondary forest soil ((-21.64 ± 4.86) μg C·m^-2·h^-1) was significantly higher than that of the artificial forest ((-10.52 ± 2.11) μg C·m^-2·h^-1). CO₂ emissions of the artificial forest soil ((106.53 ± 19.33) μg C·m^-2·h^-1) were significantly higher than that of the primary forest soil ((49.50 ± 8.16) μg C·m^-2·h^-1) and the secondary forest soil ((63.50 ± 5.35) μg C·m^-2·h^-1) (p < 0.01). N₂O emissions of the secondary forest soil ((1.91 ± 1.22) μg N·m^-2·h^-1) were higher than that of the primary forest soil ((1.40 ± 0.28) μg N·m^-2·h^-1) and the artificial forest soil ((1.01 ± 0.86) μg N·m^-2·h^-1). Reduced precipitation (-50%) had a significant inhibitory effect on CH₄ uptake of the artificial forest soil, while it enhanced CO₂ emissions of the primary forest soil and the secondary forest soil. Reduced precipitation had a significant inhibitory effect on CO₂ emissions of the artificial forest soil and N₂O emissions of the secondary forest (p < 0.01). Reduced precipitation promotes N₂O emissions of the primary forest soil and the artificial forest soil. CH₄ uptake of the primary forest and the secondary forest soil increased significantly with the increase of soil temperature under natural and reduced precipitation. CO₂ and N₂O emission fluxes of the primary forest soil, secondary forest soil and artificial forest soil were positively correlated with soil temperature (p < 0.05). Soil moisture inhibited CH₄ uptake of the secondary forest soil and the artificial forest soil (p < 0.05). CO₂ emissions of the primary forest soil were significantly positively correlated with soil moisture (p < 0.05). N₂O emissions of primary forest soil and secondary forest soil were significantly correlated with the nitrate nitrogen content (p < 0.05). It was implied that reduced precipitation and land use change would have significant effects on greenhouse gas emissions of subtropical forest soils.     

Curcumin synergizes with resveratrol to stimulate the MAPK signaling pathway in human articular chondrocytes in vitro

The mitogen-activated protein kinase (MAPK) pathway is stimulated in differentiated chondrocytes and is an important signaling cascade for chondrocyte differentiation and survival. Pro-inflammatory cytokines such as interleukin 1β (IL-1β) play important roles in the pathogenesis of osteoarthritis (OA) and rheumatoid arthritis (RA). In this study, we investigated whether curcumin and resveratrol can synergistically inhibit the catabolic effects of IL-1β, specifically the inhibition of the MAPK and subsequent apoptosis in human articular chondrocytes. Chondrocytes were either left untreated or treated with 10 ng/ml IL-1β or 1 μM U0126, a specific inhibitor of MAPK pathway alone for the indicated time periods or pre-treated with 10 μM curcumin, 10 μM resveratrol or 10 μM resveratrol and 10 μM curcumin for 4 h followed by co-treatment with 10 ng/ml IL-1β or 1 μM U0126 and 10 μM resveratrol, 10 μM curcumin or 10 μM resveratrol and 10 μM curcumin for the indicated time periods. Cultures were evaluated by immunoblotting and transmission electron microscopy. Incubation of chondrocytes with IL-1β resulted in induction of apoptosis, downregulation of β1-integrins and the extracellular signal-regulated kinase 1/2 (Erk1/2). Interestingly, U0126 induced apoptosis and blocked the above-mentioned proteins in a similar way to IL-1β. Furthermore, curcumin and resveratrol inhibited IL-1β- or U0126-induced apoptosis and downregulation of β1-integrins and Erk1/2 in human articular chondrocytes. These results suggest that combining these two natural compounds activates MEK/Erk signaling, a pathway that is involved in the maintenance of chondrocyte differentiation and survival.