Mechanism of Yanghe Pingchaun granules on airway remodeling in asthmatic rats based on IL-6/JAK2/STAT3 signaling axis

Objective: To investigate the effects of Yanghe Pingchuan Granules on airway remodeling in asthmatic rats, and to explore the mechanism of Interleukin-6/Janus kinase 2/ Signal transducing activator of transcription 3(IL-6/JAK2/STAT3) signal axis. Methods: We separated 42 healthy male SD rats into two groups, a control group (7) and a model group (35).The model group was sensitized with a combination of ovalbumin (OVA) and aluminum hydroxide for 2 weeks, while the control group was given an equal amount of physiological saline.After 2 weeks, the modeling group was randomly divided into Model group, Yanghe Pingchuan Granules high, medium and low dose groups and Dexamethasone group, each group consisted of 7 animals. After 4 weeks, OVA atomization and gavage were used for stimulation and treatment. Yanghe Pingchuan Granules high, middle and low groups were given 15.48, 7.74, 3.87 g∙kg-1 Yanghe Pingchuan Granules daily, dexamethasone group was given 0.0625 mg∙kg-1 dexamethasone daily, and the other groups were given the same amount of normal saline. HE, PAS and Masson staining were used to observe the lung histopathological changes in rats. The levels of interleukin-6, IL-23 and IL-17A were detected by ELISA. The expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 in lung tissues were detected by Western blot. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression levels of IL-6, JAK2 and STAT3 in rat lung tissue. Results: The lung tissue structure of the model group was severely damaged compared to the control group, accompanied by a great many of inflammatory cell infiltration, goblet cell hyperplasia, subepithelial collagen fiber deposition and airway epithelial thickening were more obvious. The expressions of IL-6, IL- 23 and IL-17A in serum were significantly increased (P<0.01), the protein expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 and the mRNA expression levels of IL-6, JAK2 and STAT3 in lung tissue were significantly increased (P<0.01);Compared with the model group, inflammatory cell infiltration, goblet cell proliferation, subepithelial collagen fiber deposition and airway epithelial thickening were significantly reduced in each administration group, and the expressions of IL-6, IL-23 and IL-17A in serum were significantly decreased (P< 0.01). The protein expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 and mRNA expression levels of IL-6, JAK2 and STAT3 in lung tissue were significantly decreased (P<0.01). Conclusion: Yanghe Pingchuan Granules can significantly alleviate airway remodeling in asthmatic rats, and its mechanism may be through inhibiting the IL-6/JAK2/STAT3 signal axis.

QUANTITATIVE ANALYSIS OF IL-2 AND IMMUNOMODULATING EFFECT OF IL-2 ON NK ACTIVITY OF HEPATITIS B PATIENTS

Using125I-UDR labelled K562 cells as target cells, we assay the natural killer cell （NK） activity in peripheral blood mononuclear cells （PBMCs） from 36 cases of various types of viral hepatitis B （HB）, together with 33 healthy adults as controls. At same time the NK activity was detected when PBMCs were pretreated with recombinant IL-2 （rIL-2） in 19 patients with various types of HB and 14 normal controls. We also determined the IL-2 activity produced by PBMCs in 26 HB patients and 14 normal controls. The following results were obtained: （1） The NK activity was markedly elevated in early acute hepatitis B （AH） （P【0.01）; significantly decreased in chronic active hepatitis （CAH）, chronic persistent hepatitis （CPH） and fulminant hepatitis （FH） （P【0.01）, while that of convalescents with AH was within normal range 35.85±12.52%. （2） The early rise of NK activity in acute infection and the decline in convalescence and also the parallel change in SGPT level in 3 AH cases were observed. （3） The amount of IL-2 produced by PBMCs in HB patients was lower than that of normal controls （P【0.01 ）. （4）There was no correlation between the change of IL-2 activity and NK activity in HB patients （r=0.15; P】O.05）. （5） The NK activity of most normal subjects were enhanced when the PBMCs were pretreated with rIL-2 but the latter was still within the normal ranges. These results suggest that the mechanism of the effect of IL-2 in modulating the NK activity of HB patients is very complicated. IL-2 not only directly enhances the low NK activity of HB patients, but also depresses the high NK activity. This immunomodulating effect may be influenced by serum inhibitory facts as well as the amount and the combining ability of IL-2 receptor or on NK cell surface.

Correlation of large conductance Ca2+ activated K+ channelα andβ subunit expression in uterine smooth muscle with the postpartum hemorrhage induced by uterine inertia

Objective:To study the correlation of large conductance Ca2+ activated K+ channel (BKCa)α andβ subunit expression in uterine smooth muscle with the postpartum hemorrhage induced by uterine inertia.Methods: The puerperae who underwent cesarean section and had postpartum hemorrhage induced by uterine inertia in Panzhihua Women and Children Health Hospital between March 2015 and May 2017 were selected as the hemorrhage group of the study, and the puerperae who underwent cesarean section and were without postpartum hemorrhage in Panzhihua Women and Children Health Hospital during the same period were selected as the control group. Proper amount of uterine muscle tissue was collected during the cesarean section to measure the expression of BKCaα andβ subunits and the levels of contraction-related proteins in uterine muscle as well as the contraction characteristic parameters of the uterine muscle.Results: The mRNA expression and protein expression of BKCaα andβ subunits in uterine muscle tissue of hemorrhage group were significantly higher than those of control group;the contraction amplitude, contraction frequency and contraction activity of uterine muscle tissue as well as the OTR, COX2, CX43 and HSP27 levels in uterine muscle tissue of hemorrhage group were significantly lower than those of control group;the BKCaα andβ subunit expression in uterine muscle tissue of hemorrhage group were negatively correlated with the contraction amplitude, contraction frequency and contraction activity as well as the OTR, COX2, CX43 and HSP27 levels.Conclusion: The high expression of BKCa in uterine smooth muscle can reduce the uterine muscle contractility and decrease the levels of contraction-related proteins, and it is closely related to the occurrence of postpartum hemorrhage induced by uterine inertia.

基于Hepcidin和JAK2/STAT3信号通路探讨通痹颗粒 对胶原诱导性关节炎大鼠的影响

目的研究通痹颗粒对胶原诱导性关节炎(collagen-induced arthritis,CIA)大鼠铁调素(hepcidin,Hepc)、Janus激酶(janus kinase,JAK)2/信号转导子和转录激活子(signal transduction and activator of transcription,STAT)3信号通路的影响。方法选取36只雌性SD大鼠随机分成空白组、模型组、阳性对照组和通痹颗粒低、中、高剂量组,每组6只。空白组不予处理,其余组用牛Ⅱ型胶原建立CIA模型。造模完成后,空白组、模型组予生理盐水灌胃,其余各组分别以巴瑞替尼片和低、中、高剂量通痹颗粒灌胃。每天1次,连续4周。HE染色行滑膜组织病理学观察;酶联免疫吸附法测定血清Hepc、白细胞介素6(interleukin 6,IL-6)水平;逆转录-聚合酶链反应法测定滑膜中JAK2、STAT3、细胞信号因子传导抑制体(suppressor of cytokine signaling,SOCS)1、SOCS3的mRNA相对表达量;Western blot法检测滑膜中JAK2、p-JAK2、STAT3、p-STAT3、SOCS1、SOCS3的蛋白表达量。结果模型组见滑膜上皮结构缺损,滑膜重度增生,排列紊乱,并有大量炎症细胞浸润和多个血管翳形成;各给药组滑膜炎症均有所减轻,阳性对照组优于通痹颗粒高剂量组,通痹颗粒中、高剂量组优于低剂量组。与模型组相比,各给药组关节炎指数评分、血清Hepc和IL-6水平均显著降低(P<0.01);与阳性对照组相比,通痹颗粒中、低剂量组关节炎指数评分、血清Hepc和IL-6水平均升高(P<0.05)。与模型组比较,阳性对照组和通痹颗粒低、中、高剂量组JAK2、STAT3 mRNA和蛋白以及p-JAK2、p-STAT3的蛋白表达量均降低(P<0.05),而通路抑制因子SOCS1、SOCS3 mRNA和蛋白的表达均升高(P<0.05);与阳性对照组比较,通痹颗粒各剂量组JAK2、STAT3 mRNA和蛋白以及p-JAK2、p-STAT3的蛋白表达量均升高(P<0.05),而SOCS1、SOCS3 mRNA和蛋白的表达均降低(P<0.05)。结论通痹颗粒能够改善CIA大鼠滑膜炎症,其机制可能与抑制JAK2/STAT3信号通路而减少Hepc的表达有关。

Anti-tumor activities of macromolecular fractions of fresh gecko vivo and their induction of Bel-7402 cell differentiation

Objective:To investigate the anti-tumor effect of macromolecular fractions of fresh gecko (M-AG) in vivo and their differentiation-inducing activity in Bel-7402 cells in vitro.Methods:An H22 hepatocarcinoma-bearing mouse model was used to evaluate the anti-tumor activity of M-AG samples.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was applied to analyze cell viability.Cell morphology was observed by phase contrast microscopy.The quantity of the alpha-fetoprotein was detected by a radioimmunoassay.Chromatometry was used to assay the albumin quantity.Activities of alkaline phosphatase and γ-glutamyl trans-peptidase were measured by biochemical methods.Finally,western blotting was applied to assess proteins in the mitogen-activated protein kinase (MAPK) signaling pathway.Results:Macromolecular fractions of fresh gecko exerted a significant anti-tumor effect in mice.The inhibition rate of tumor growth was 63% in the moderate M-AG dose group.Cells treated with M-AG displayed a differentiated state.The treatment lowered alphafetoprotein secretion and significantly decreased the activities of γ-glutamyl trans-peptidase and alkaline phosphatase in Bel-7402 cells.In contrast,M-AG increased the amount of albumin in the cell culture medium.All biochemical indices demonstrated that M-AG induced Bel7402 cell differentiation.Western blotting showed no changes in the quantities of extracellular signal-regulated kinase (ERK) 1/2,p38MAPK,or c-Jun N-terminal protein kinase 1/2.However,M-AG significantly activated the phosphorylation of ERK1/2 in a dose-dependent manner.In addition,M-AG had no significant influence on the expression of nuclear factor-kappa B.Conclusion:Macromolecular fractions of fresh gecko has an anti-tumor activity in H22 hepatocarcinoma-bearing mice in vivo and inhibits Bel-7402 cell proliferation in vitro by inducing cell differentiation related to activation of ERK1/2.

The fertilization-induced Ca^(2+) oscillation in mouse oocytes is cytoplasmic maturation dependent

Mature eggs (at metaphase Ⅱ stage) produce a series of Ca2+ oscillation at fertilization. To define whether the fertilization-induced Ca2+ oscillation is restrict to the metaphase Ⅱ eggs and cell cycle dependent, mouse oocytes at prophase Ⅰ (arrested at germinal vesicle stage),metaphase Ⅰ, metaphase Ⅱ, as well as the pronuclear embryos at interphase of the first mitotic division derived from fertilization or parthenogenetic activation were inseminated after removal of zona pellucida. The results show that the fertilization-induced Ca2+ oscillation is not specific to metaphase Ⅱ eggs. This is supported by the fact that immature oocytes generated the Ca2+ oscillations at fertilization regardless of their nuclear progression from prophase Ⅰ to metaphase Ⅰ (in vitro matured) stage. More interestingly, it was first found that pronuclear embryos at interphase derived from parthenogenetic activation showed Ca2+ oscillations in response to fertilization while the zygotes at interphase did not after reinsemination or intracytoplasmic injection of sperm extracts which induce Ca2+ oscillations in MII eggs. This suggests that the ability of oocytes to generate Ca2+ oscillation in response to sperm penetration is not regulated in a cell cycle dependent manner but dependent on the cytoplasmic maturation.

Measurement of the Activation of IL-2 on Bone Marrow by Flow Cytometry

The changes of cell surface markers before and after activation by IL-2were detected by flow cytometry (FCM) to establish a more convenient and pre-cise criterion for the judgment of the activation of bone marrow. By using the measurement of the release of lactate dehydrogenase (LDH) the cytotoxicity of mononuclear cells (MNCs) from bone marrow, activated or inactivated, on tumor cell line K562 was evaluated, and at the same time the changes of surface markers on MNCs before and after activation were examined by using FCM. The results showed that the cytotoxicity of MNCs from bone marrow activated by IL-2 on tu-mor cell line K562 was increased obviously and the number of CD and CD posi-tive cells in bone marrow MNCs was higher than be fore activation. The enhanced cytotoxicity of MNCs on tumor cell line K562 was synchronous with the increaseof the number of CD and CD positive cells in 48 to 72 h. It is more direct, simple and precise to demonstrate the activation of IL-2 on bone marrow by detecting the changes of the amount of the CD and CD positive cells in bone marrow by FCM.

芒柄花素调节JAK2/STAT3信号通路对妊娠高血压大鼠的治疗作用

目的:探讨芒柄花素(FMN)对妊娠高血压(PIH)大鼠的治疗作用及可能机制。方法:将妊娠大鼠分为正常组(Normal组,生理盐水)、PIH组(生理盐水)、FMN组[40 mg/(kg·d)FMN]、Janus激酶2(JAK2)抑制剂组[AG490组,5 mg/(kg·d)AG490]、FMN+JAK2抑制剂组[FMN+AG490组,40 mg/(kg·d)FMN+5 mg/(kg·d)AG490],每组12只。除Normal组外,其余各组大鼠连续4 d皮下注射125 mg/(kg·d)L-精氨酸甲酯构建PIH模型。观察大鼠尾静脉压、24 h尿蛋白含量,血清血管内皮因子[内皮素1(ET-1)、一氧化氮(NO)]、炎性因子[肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)]、心肌损伤指标[肌酸激酶同工酶MB(CK-MB)、心肌肌钙蛋白I(cTnI)]、肾损伤指标[血尿素氮(BUN)、血肌酐(SCr)]水平,心肌、肾组织病理学变化及心肌、肾组织中氧化应激指标[丙二醛(MDA)和总超氧化物歧化酶(T-SOD)]和JAK2/信号转导和转录激活因子3(STAT3)信号通路相关蛋白(JAK2、p-JAK2、STAT3、p-STAT3)表达。结果:给药结束后,与Normal组比较,PIH组大鼠尾静脉压、24 h尿蛋白含量升高,血清ET-1、TNF-α、IL-6、CK-MB、cTnI、BUN、SCr水平及心肌、肾组织中MDA含量和p-JAK2/JAK2、p-STAT3/STAT3比值升高(P<0.05),血清NO水平和心肌、肾组织中T-SOD活性降低(P<0.05),且心肌、肾组织存在明显的病理损伤。与PIH组比较,FMN组、AG490组和FMN+AG490组大鼠尾静脉压、24 h尿蛋白含量降低,血清ET-1、TNF-α、IL-6、CK-MB、cTnI、BUN、SCr水平及心肌、肾组织中MDA含量和p-JAK2/JAK2、p-STAT3/STAT3比值降低(P<0.05),血清NO水平和心肌、肾组织中T-SOD活性升高(P<0.05),且心肌、肾组织病理损伤均有所改善;其中FMN+AG490组上述指标变化均显著优于FMN组、AG490组。结论:FMN可能通过抑制JAK2/STAT3信号通路,减轻心肌、肾组织中氧化应激和全身炎症反应,改善血管内皮舒缩功能,降低血压,减轻PIH大鼠心肌、肾组织损伤。

IL-2和IL-15的生物学及免疫治疗应用研究进展

IL-2和IL-15对于调控体内淋巴细胞功能和稳态起重要作用。对二者及其受体细胞生物学和分子生物学的认识为癌症免疫治疗提供了应用前景。二者在体外具有类似的刺激淋巴细胞增殖作用,然而在体内的作用却明显不同。不同生理功能是由于各自不同受体亚基介导的不同信号转导通路、不同受体亚基表达模式。最近研究发现反式呈递方式介导的信号转导是IL-15功能不同于IL-2的一个重要机制。尽管它们的异三聚体受体中有两个共用亚基,这两种细胞因子在适应性免疫应答中具有明显不同的作用。IL-2独特的作用是清除自身反应性T细胞防止自身免疫性疾病发生,相反,IL-15可长久维持记忆性T细胞针对病原体的免疫应答。本文将就癌症和自身免疫性疾病治疗中涉及的IL-2和IL-15二种细胞因子生物学作一综述。

氦氖激光合用环磷酰胺对S180荷瘤鼠脾脏淋巴细胞IL-2诱生活性和增殖反应的影响

以S180腹水瘤小鼠为动物模型,应用11.00、14.67和22.00J/cm23种剂量氦氖激光照射荷瘤鼠哈德腺,同时联合应用化疗药物环磷酰胺,对S180荷瘤鼠脾脏淋巴细胞IL-2诱生活性和淋巴细胞增殖反应的影响作了系统地研究。结果表明:CYT/氦氖激光照射联合应用组IL-2诱生活性和淋巴细胞增殖反应均明显高于CYT对照组。CYT可对荷瘤体产生某种程度的免疫抑制效应,而氦氖激光照射则可在某种程度上改善这一效应。

毒害艾美耳球虫二次感染对雏鸡免疫器官IL-2诱生活性及淋巴细胞增殖功能的影响

应用细胞培养技术结合MTT检测法，通过白细胞介素-2（IL-2）诱生活性和T、B淋巴细胞对ConA或PMA增殖能力的检测，研究雏鸡二次攻击性感染毒害艾关耳球虫后，其胸腺、脾脏和法氏囊上述被检指标的动态变化。结果发现，雏鸡二次攻击性感染毒害艾美耳球虫后，其胸腺、脾脏T淋巴细胞IL-2诱生活性在感染后4～7d较对照和一次感染雏鸡显著降低，7d后迅速回升，且幅度较大。T、B淋巴细胞对ConA或PMA的增殖功能在二次感染后的早期下降，随后也迅速回升。

PREPARATION AND CYTOLYTIC TUMOR ACTIVITY OF OSTEOSARCOMA TUMOR INFILTRATING LYMPHOCYTES IN VITRO

In this study, the isolation, purification and differentiation of tumor-lnflltratlng lymphocytes (TIL) from 6 fresh osteosarcoma specimens were achieved by discontinuous density gradient centrifugation. One specimen of the osteosarcoma TIL were enlarged in IL-2 for long time in vitro, reaching 28 days and their cytolytic activity against different tumor cell lines was Investigated. The experimental results indicated that the preparation of osteosarcoma TIL adopted by the mechanical means was simple, having higher purifity, keeping higher effects on killing NK- sensitive tumor cell lines and NK-insensitive tumor cell lines as well as rapid proliferation in vitro cultured in IL-2.

微波诱导下2-芳氧甲基苯并咪唑-1-水杨醛乙酰腙衍生物合成及生物活性研究

在微波辐射条件下,通过2-芳氧甲基苯并咪唑-1-乙酰肼衍生物与水杨醛进行缩合反应,合成了10种未见文献报道的2-芳氧甲基苯并咪唑-1-水杨醛乙酰腙衍生物.它们的结构经元素分析,IR,~1H NMR和^(13)C NMR确证.初步生物活性试验结果表明该系列化合物对小麦幼苗的生长具有明显的调节作用.

骨形态发生蛋白-2分离纯化技术研究进展

骨形态发生蛋白-2(bovine bone morphogenetic protein,BMP-2)是骨基质分泌的能诱导骨髓基质细胞(Bone Marrow Stem Cells,BMSCs)定向分化和异位成骨的生长因子,临床应用前景广阔。BMP-2可以通过天然牛骨中和基因重组诱导表达后分离纯化两种方法制备,受到天然牛骨中含量过少和活性提取的限制,主要采用基因重组方法。本文系统地介绍了BMP-2分离纯化方法的研究进展。

苦参碱对妊娠高血压大鼠内皮损伤和JAK2/STAT3/SOSC1信号通路的影响

目的探讨苦参碱对妊娠高血压(PIH)大鼠内皮损伤及酪氨酸蛋白激酶2/信号转导子和转录激活子3/细胞因子信号转录抑制因子1(JAK2/STAT3/SOSC1)信号通路的影响。方法将60只孕鼠随机分成正常对照组、模型对照组、低剂量苦参碱组、高剂量苦参碱组、硫酸镁组,每组12只。正常对照组之外的各组孕鼠在妊娠第12天通过灌胃50 mg/kg亚硝基左旋精氨酸甲酯构建PIH大鼠模型。在妊娠第16天,低、高剂量苦参碱组分别灌胃50、100 mg/kg苦参碱,硫酸镁组灌胃100 mg/kg硫酸镁,正常对照组和模型对照组则灌胃等体积的生理盐水。分别使用大鼠无创血压计和考马斯亮蓝法检测各组孕鼠妊娠第16天(给药前)、第17天、第21天的尾动脉血压及24 h尿蛋白含量;酶联免疫分析试剂盒检测血清中超氧化物歧化酶(SOD)、丙二醛(MDA)、肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-6、IL-10、内皮素(ET)、血栓素B2(TXB2)、一氧化氮(NO)、6酮前列腺素F1α(6-keto-PGF1α)水平;蛋白免疫印迹法检测大鼠胎盘组织中JAK2、p-JAK2、STAT3、p-STAT3、SOSC1蛋白表达水平。结果妊娠第17、21天,模型对照组孕鼠血压、24 h尿蛋白含量明显高于正常对照组(P<0.05),低、高剂量苦参碱组孕鼠血压、24 h尿蛋白含量明显比模型对照组低(P<0.05)。与正常对照组比较,模型对照组大鼠血清中SOD、IL-10、NO和6-keto-PGF1α水平降低,MDA、TNF-α、IL-6、ET和TXB2以及胎盘组织中p-JAK2/JAK2、p-STAT3/STAT3、SOSC1蛋白水平升高(P<0.05);与模型对照组相比,低、高剂量苦参碱组SOD、IL-10、NO和6-keto-PGF1α依次增加,MDA、TNF-α、IL-6、ET和TXB2以及胎盘组织中p-JAK2/JAK2、p-STAT3/STAT3、SOSC1蛋白水平依次降低(P<0.05);与硫酸镁组相比,高剂量苦参碱组上述指标均无明显改变(P>0.05)。结论苦参碱降低PIH模型大鼠血压、尿蛋白含量、炎症反应及氧化应激水平,抑制JAK2、STAT3磷酸化及SOSC1蛋白表达,进而缓解PIH大鼠内皮损伤。

髓芯减压联合骨形成蛋白活性诱导棒植入术治疗早期股骨头坏死的效果

目的探讨髓芯减压联合骨形成蛋白(BMP)活性诱导棒植入术治疗早期股骨头坏死的效果。方法回顾性分析2018年6月至2022年6月收治的116例早期股骨头坏死患者,按照不同的手术方式分为髓芯减压联合BMP活性诱导棒组(BMP组)和同种异体骨组。BMP组60例,采用髓芯减压联合BMP活性诱导棒植入术治疗。同种异体骨组56例,采用髓芯减压联合同种异体骨打压植骨术治疗。对比两组患者术前、术后6个月和术后1年的髋关节Harris评分、疼痛视觉模拟评分(VAS)的差异,并对患者术后1年疗效和股骨头存活率进行比较。结果所有患者均获得随访,两组患者术前VAS评分、Harris评分差异无统计学意义(P>0.05),术后6个月、1年时两组患者VAS评分、Harris评分均显著改善,且BMP组优于同种异体骨组,差异有统计学意义(P<0.05)。术后1年时,BMP组Harris髋关节评分优良率高于同种异体骨组,差异有统计学意义(P<0.05);BMP组股骨头存活率高于同种异体骨组,差异有统计学意义(P<0.05)。结论髓芯减压联合BMP活性诱导棒植入术治疗早期股骨头坏死早期具有疗效,能够加速诱导新骨生成,提高新骨质量,为股骨头提供生物力学支撑,有效避免股骨头塌陷,且生物相容性好、能够在体内降解吸收,值得在临床上推广。

二甲双胍减缓2型糖尿病小鼠非酒精性脂肪肝的作用

目的探讨二甲双胍对2型糖尿病(type 2 diabetes mellitus,T2DM)小鼠非酒精性脂肪肝(non alcohol fatty liver disease,NAFLD)的减缓作用及其机制。方法将20只2型糖尿病db/db小鼠随机分成糖尿病模型组和二甲双胍组,另选10只同周龄正常db/m小鼠为正常对照组。糖尿病模型组和正常对照组灌胃蒸馏水,二甲双胍组灌胃二甲双胍(0.16 g/kg),干预8周后处死取肝脏组织,观察组织形态学改变。免疫组化法和Western Blot法检测肝脏组织中腺苷酸活化蛋白激酶α1(AMP-activated protein kinaseα1,AMPKα1)、低氧诱导因子-1α(hypoxia inducible factor 1α,HIF-1α)、乙酰辅酶A羧化酶(acetyl coa carboxylase,ACC)的表达情况。结果糖尿病模型组出现弥漫性肝细胞大泡性脂肪变,二甲双胍组肝细胞脂肪变性情况较糖尿病模型组明显减轻,且AST、ALT水平明显降低(均P <0.01)。二甲双胍组AMPKα1蛋白表达水平和AMPKα1、ACC磷酸化水平明显高于其他组,而ACC、HIF-1α蛋白表达水平明显低于其他组(均P <0.01)。结论二甲双胍可能通过激活AMPK信号通路,促进ACC蛋白磷酸化,抑制HIF-1α进而达到减缓2型糖尿病小鼠NAFLD发生的作用。

磷脂酰肌醇-3-激酶/蛋白激酶B和丝裂原活化蛋白激酶/细胞信号调节激酶1/2信号通路抑制剂对乳腺癌细胞缺氧诱导因子-2α表达的影响

目的探讨磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/Akt)和丝裂原活化蛋白激酶/细胞信号调节激酶1/2(MEK/ERK1/2)信号通路抑制剂对缺氧诱导的乳腺癌MCF-7细胞中缺氧诱导因子-2α(HIF-2α)表达的影响。方法乳腺癌MCF-7细胞常规培养24 h后,分为常氧组、缺氧组、缺氧+低剂量LY294002组、缺氧+高剂量LY294002组,缺氧+低剂量U0126组和缺氧+高剂量U0126组。常氧组细胞使用含生理盐水的RPMI 1640培养基,缺氧组细胞使用含100μmol·L-1氯化钴的RPMI 1640培养基,缺氧+低剂量LY294002组细胞使用含100μmol·L-1氯化钴和4μmol·L-1 LY294002的RPMI 1640培养基,缺氧+高剂量LY294002组细胞使用含100μmol·L-1氯化钴和40μmol·L-1 LY294002的RPMI 1640培养基,缺氧+低剂量U0126组细胞使用含100μmol·L-1氯化钴和2μmol·L-1 U0126的RPMI 1640培养基,缺氧+高剂量U0126组细胞应用含8μmol·L-1 U0126的RPMI 1640培养基,继续培养3 h。采用实时荧光定量聚合酶链式反应检测各组细胞HIF-2αmRNA的表达;采用Western blot法检测各组细胞HIF-2α蛋白、磷酸化Akt(p-Akt)和磷酸化ERK1/2(p-ERK1/2)蛋白的表达。结果缺氧组细胞中HIF-2αmRNA相对表达量及HIF-2α蛋白、p-Akt蛋白和p-ERK1/2蛋白相对表达量高于常氧组(P<0.05);缺氧+低剂量LY294002组、缺氧+高剂量LY294002组、缺氧+低剂量U0126组、缺氧+高剂量U0126组细胞中HIF-2αmRNA相对表达量及HIF-2α蛋白、p-Akt蛋白和p-ERK1/2蛋白相对表达量低于缺氧组(P<0.05)。缺氧+高剂量LY294002组细胞中HIF-2αmRNA相对表达量及HIF-2α蛋白、p-Akt蛋白和p-ERK1/2蛋白相对表达量低于缺氧+低剂量LY294002组(P<0.05);缺氧+高剂量U0126组细胞中HIF-2αmRNA相对表达量及HIF-2α蛋白、p-Akt蛋白和p-ERK1/2蛋白相对表达量低于缺氧+低剂量U0126组(P<0.05)。结论PI3K/Akt和MEK/ERK1/2信号通路可能是缺氧诱导HIF-2α表达的上游信号通路,在缺氧条件下对HIF-2α具有正向调节作用,在乳腺癌的发生、发展中发挥重要作用。

人骨形成蛋白-2C端102肽的诱骨活性

为分析更短的hBMP 2C端肽是否具有诱骨活性 ,寻求新型的有诱骨活性的基因工程hBMP 2产品。利用温度诱导的大肠杆菌表达系统表达肽段长度为 10 2个氨基酸的hBMP 2C端肽及其Cys的突变体。表达产物经纯化复性后 ,植入小鼠肌袋模型中测试其诱骨活性。获得了能稳定表达hBMP 2C端肽的工程菌 ,测序结果与预期的序列完全一致。表达产物以包涵体形式存在 ,表达量占细胞总蛋白的 3 0 %。产物经纯化复性后 ,小鼠肌袋模型测试结果表明 :hBMP 2 10 2肽仍具有诱骨活性 ,而将C端第一位Cys突变的 10 2肽诱骨活性丧失。实验表明 :比hBMP 2成熟肽 ( 114个氨基酸 )更短的C端 10 2肽仍具有良好诱骨活性 ,这 10 2肽N端第一个Cys对其诱骨活性可能是必需的。

基于抗炎活性的香花崖豆藤化学成分研究

目的分离香花崖豆藤中发挥抗炎活性的化学成分。方法香花崖豆藤70%乙醇冷浸提取物经聚酰胺柱梯度洗脱,得水洗脱部位(A)、40%乙醇洗脱部位(B)、70%乙醇洗脱部位(C)、100%乙醇洗脱部位(D)。采用凝胶色谱、半制备高效液相色谱对抗炎有效部位进行化学成分分离。以脂多糖(LPS)诱导的RAW264.7细胞为炎性模型,试验分为A组、B组、C组、D组(50μg/mL极性洗脱部位A,B,C,D),阳性对照组(0.5μmol/L地塞米松),模型组(10μg/mL LPS),空白组(常规培养基)。采用Griess法测定一氧化氮(NO)释放量;采用免疫印迹(Western blot)法测定诱导型一氧化氮合酶(iNOS)、环氧合酶2(COX-2)的蛋白表达水平,筛选抗炎有效部位。结果香花崖豆藤40%乙醇洗脱部位经分离得到2个单体化合物,分别鉴定为5,7-dihydroxy-2',3',4'-trimethoxyisoflavanone(化合物1)和5,7,4'-trihydroxyiso flavanone(化合物2)。与模型组比较,B组、C组、D组的NO释放量均显著减少(P<0.001),B组的iNOS蛋白表达水平显著降低(P<0.01)。结论香花崖豆藤40%乙醇洗脱部位具有较好的抗炎活性,可通过抑制iNOS蛋白的表达而发挥抗炎作用。