参榆洗液改善痔术后大鼠痛觉敏化及对cAMP/PKA信号通路的影响

目的:基于环磷酸腺苷/蛋白激酶A(c AMP/PKA)信号通路探究参榆洗液对痔术后大鼠疼痛的影响。方法:构建痔术后大鼠模型,将造模成功的56只SD大鼠随机分为模型组、溶剂模型组、参榆洗液组、参榆洗液+Forskolin(c AMP/PKA信号通路激动剂)组、Forskolin组、H-89(c AMP/PKA信号通路抑制剂)组、NS-398(PGE_(2)抑制剂)组,每组8只;另取8只大鼠为空白组。各组大鼠给予对应药物干预,连续7 d。给药结束后1 d,观察大鼠一般状态及疼痛相关动物学行为,观察创面变化并计算创面愈合率,苏木精-伊红(HE)染色观察各组大鼠创面组织病理学改变,蛋白质免疫印迹法(Western blotting)检测各组大鼠背根神经节TRPV1、p-PKA蛋白表达及创面组织中c AMP、PKA蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠创面组织中c AMP m RNA、PKA m RNA表达,酶联免疫吸附测定法(ELISA)检测创面组织中PGE_(2)、IL-6、IL-1β含量。结果:模型组大鼠背根神经节组织中p-PKA、TRPV1及创面周围组织c AMP、PKA、PGE_(2)、c AMP m RNA、PKA m RNA、IL-6、IL-1β表达水平升高(P<0.05);与模型组比较,H-89组、NS-398组、参榆洗液+Forskolin组、参榆洗液组创面组织病变程度减轻,创面愈合率依次上升,背根神经节组织中p-PKA、TRPV1及创面组织c AMP、PKA、c AMP m RNA、PKA m RNA、IL-6、PGE_(2)、IL-1β表达水平依次降低(P<0.05);Forskolin干预能显著提高痔术后大鼠模型c AMP、PKA、TRPV1、p-PKA、PGE_(2)、IL-6、IL-1β表达水平(P<0.05),与参榆洗液联合干预后,上述蛋白及炎症因子表达水平显著下降(P<0.05)。结论:参榆洗液对痔术后大鼠cAMP/PKA信号通路有抑制作用,抑制c AMP/PKA信号通路可改善痔术后痛觉敏化,其机制可能与抑制PGE_(2)表达、减少炎症因子释放、抑制TRPV1活化有关。

Effect of Down-regulation of MMP-2 by Quercetin on the Migration and Invasion of Uterine Fibroids Cells through the cAMP Signaling Pathway

Objective:To investigate the effect of down-regulation of matrix metalloproteinase-2(MMP-2)by down-regulating the cyclic adenosine monophosphate(cAMP)signaling pathway on the migration and invasion of uterine fibroids cells.Methods:A total of 65 female SD rats were randomly divided into five groups after being adaptively fed for 7 d.Except for the blank group,the remaining four groups of rats were injected with 0.05 mg/(100 g·d)estradiol benzoate and 0.5 mg/(100g·d)progesterone into the muscle to establish a uterine fibroids rat model.After successful modeling,the blank group and model group were given 200 mg/(kg·d)physiological saline by gavage,the low-dose group was given 100 mg/(kg·d)mifepristone by gavage,the medium-dose group was given 200 mg/(kg·d)mifepristone by gavage,and the high-dose group was given 300 mg/(kg·d)mifepristone by gavage.After continuous gavage treatment for 21 d,serum and uterine tissues were collected from rats to observe and compare the expression levels of MMP-2 mRNA,TIMP-2 mRNA,estradiol(E2),cAMP signaling pathway related proteins,and the migration and invasive ability of uterine fibroids cells in the five groups of rats.Results:The expression levels of MMP-2 mRNA and E2in the high-dose group were lower than those in the low-dose group and the medium-dose group,and the expression level of TIMP-2 mRNA was higher than that in the low-dose group and the medium-dose group(P<0.05).The number of cell migration in a single visual field of rats in the high-dose group was lower than that in the low-dose group and the medium-dose group(P<0.05).The number of cell invasion in a single visual field of rats in the high-dose group was lower than that in the low-dose group and the medium-dose group(P<0.05).The mRNA expression levels of cAMP,PDE and PK in the high-dose group were higher than those in the low-dose group,and the mRNA expression levels of AC in the high-dose group were lower than those in the low-dose group(P<0.05).The mRNA expression levels of cAMP and PDE in the high-dose group were higher than those in the mediumdose group(P<0.05).Conclusion:Letrozole may down-regulate the expression level of MMP-2 through the cAMP signaling pathway to inhibit the migration and invasion of uterine fibroids cells.With increasing doses of letrozole,its inhibitory effect on the migration and invasion of uterine fibroids cells will be enhanced.However,the optimal dosage of letrozole for inhibiting the migration and invasion of uterine fibroids cells is yet to be determined.

CCR2可能通过cAMP-PKA信号通路参与大鼠炎性疼痛

目的：探讨趋化因子受体2（chemokine receptor 2,CCR2）和c AMP-PKA信号通路在大鼠炎性疼痛发生及维持中的作用。方法：72只雌性大鼠随机分为假手术组,炎性疼痛组和CCR2抑制剂组,每组24只,各组大鼠分别采取不同处理措施,分别于处理前（T0）、注药后1 d（T1）、3 d（T2）、5 d（T3）和7 d（T4）时,对左侧后足底双足机械缩爪阈值（mechanical paw withdrawal threshold,MWT）进行测定;分别于处理后T2、T3和T4时,利用ELISA法测定背根神经节细胞中c AMP水平,利用荧光定量PCR检测CCR2、PKA和CREB在背根神经节中的表达,利用Western blot检测p CREB蛋白在背根神经节中表达。结果：与T0时相比,炎性疼痛组和CCR2抑制剂组大鼠T1-4时MWT均降低,差异均有统计学意义（P〈0.05）;炎性疼痛组和CCR2抑制剂组大鼠T1-4时MWT均低于假手术组,CCR2抑制剂组大鼠T1-4时MWT均高于炎性疼痛组,差异均有统计学意义（P〈0.05）;炎性疼痛组和CCR2抑制剂组大鼠T2-4时c AMP、CCR2 m RNA、PKA m RNA、CREB m RNA及p CREB蛋白表达量均高于假手术组,CCR2抑制剂组大鼠T2-4时c AMP、CCR2 m RNA、PKA m RNA、CREB m RNA及p CREB蛋白表达量均低于炎性疼痛组,差异均有统计学意义（P〈0.05）。结论：背根神经节中CCR2在炎性疼痛发生及进展中发挥重要作用,CCR2可能通过c AMP-PKA信号通路而参与炎性疼痛的发生。

来曲唑通过cAMP-PKA信号通路影响子宫肌瘤细胞中MMP-2和TIMP-2的表达

目的探讨cAMP-PKA信号通路在来曲唑影响子宫肌瘤细胞中MMP-2和TIMP-2表达中的作用。方法cAMP-PKA信号通路抑制剂H-89(10μmol/L)处理原代培养的人子宫肌瘤细胞30 min后,来曲唑(10-8、10-7、10-6和10-5mol/L)处理24、48和72 h。采用实时定量PCR和Western blot分别检测MMP-2和TIMP-2 mRNA和蛋白的表达。结果来曲唑以浓度和时间依赖的方式下调了人子宫肌瘤细胞中MMP-2 mRNA和蛋白的表达,上调了TIMP-2 mRNA和蛋白的表达(P均<0.05)。H-89部分取消了来曲唑诱导的人子宫肌瘤细胞中MMP-2表达的下调和TIMP-2表达的上调(P均<0.05)。结论来曲唑通过cAMP-PKA信号通路影响子宫肌瘤细胞中MMP-2和TIMP-2的表达。

利拉鲁肽通过cAMP/PKA/CREB通路干预2型糖尿病大鼠骨质疏松的机制研究

目的:探讨利拉鲁肽通过环磷酸腺苷(cAMP)/蛋白激酶A(PKA)/cAMP反应元件结合蛋白(CREB)通路干预2型糖尿病大鼠骨质疏松的机制。方法:建立2型糖尿病骨质疏松大鼠模型,随机分为模型组、利拉鲁肽组、N-[2-(对溴甲酰氨基)乙基]-5-异喹啉磺酰胺·2HCl水合物(H-89,PKA抑制剂)组和利拉鲁肽+H-89组,每组12只;另取12只SD大鼠设为假手术组。分组处理后,通过微计算机断层扫描术(micro-CT)检测大鼠股骨干骺端显微结构,比较骨矿物质密度(BMD)、骨小梁厚度(Tb.Th)、骨体积分数(BV/TV)和骨小梁数量(Tb.N);通过骨科生物力学测试评估大鼠股骨生物力学状况,比较最大载荷、屈服载荷和弹性模量;检测大鼠股骨矿盐含量;通过试剂盒检测大鼠血清活性氧(ROS)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)和cAMP水平;通过Western blot法检测大鼠骨组织cAMP/PKA/CREB通路相关蛋白(p-PKA/PKA和p-CREB/CREB)水平。体外培养大鼠股骨成骨细胞,随机分为对照组、模型组、利拉鲁肽组、H-89组和利拉鲁肽+H-89组,后3组以16.5 mmol/L葡萄糖处理以构建细胞模型。分组处理后,通过CCK-8实验检测细胞活力;通过试剂盒检测细胞cAMP水平;通过Western blot法检测细胞cAMP/PKA/CREB通路相关蛋白(p-PKA/PKA和p-CREB/CREB)水平。结果:与假手术组(对照组)相比,模型组大鼠BMD、BV/TV、Tb.N、Tb.Th、最大载荷、屈服载荷、弹性模量、骨矿盐含量,血清CAT、GSH-Px及cAMP水平,细胞活力、细胞cAMP水平、骨组织及细胞p-PKA/PKA和p-CREB/CREB水平显著降低(P<0.05),ROS和MDA水平显著升高(P<0.05)。与模型组及利拉鲁肽+H-89组分别相比,利拉鲁肽组大鼠BMD、BV/TV、Tb.N、Tb.Th、最大载荷、屈服载荷、弹性模量、骨矿盐含量,血清CAT、GSH-Px及cAMP水平,细胞活力、细胞cAMP水平、骨组织及细胞p-PKA/PKA和p-CREB/CREB均显著升高(P<0.05),ROS和MDA水平均显著降低(P<0.05);H-89组上述指标均呈相反改变(P<0.05)。结论:利拉鲁肽可激活cAMP/PKA/CREB信号通路,进而清除高糖诱导的ROS,降低2型糖尿病大鼠氧化应激水平,提高其骨生物力学性能及骨矿化,缓解骨流失,减轻大鼠骨质疏松症状。

GRK2 overexpression inhibits IGF1-induced proliferation and migration of human hepatocellular carcinoma cells by down-regulating EGR1

OBJECTIVE To investigate the role and mechanism of G protein-coupled receptor kinase 2(GRK2)involving in hepatocel ular carcinoma(HCC)progression.METHODS Cel Counting Kit 8 and tumor colony formation assay were designed to detect HCC cell proliferation,wound healing assay was to detect HCC migration.The correlation between GRK2 and early growth response-1(EGR1)were detected by RT-PCR and real-time PCR assays.Co-immunoprecipitation and Western blot assay were adopted to detect the relationship between GRK2and insulin-like growth factor 1 receptor(IGF-1R)signaling pathway.RESULTS In this study we find that GRK2plays an inhibition role in IGF1-induced HCC cell proliferation and migration.Overexpression of GRK2 causes a decrease in EGR1 expression,while knockdown of GRK2 leads to the dramatically increase in EGR1 expression in the treatment of IGF1.Through co-immunoprecipitation and Western blot assay,we confirm that GRK2can interact with IGF-1R and inhibiting IGF1-induced activation of IGF1R signaling pathway.Silencing EGR1attenuates GRK2 overexpression-caused inhibition of cell proliferation,tumor colony number and migrationactivity,while overexpressing of EGR1 restores the antiproliferative and migratory effect by GRK2 overexpression in HCCLM3 cells.CONCLUSION Taken together,these results suggest that GRK2 may inhibit IGF1-induced HCC cell growth and migration through down-regulation of EGR1 and indicate that enforced GRK2 may offer a potential therapeutic approach against HCC.

Rbm8a regulates neurogenesis and reduces Alzheimer's disease-associated pathology in the dentate gyrus of 5×FAD mice

Alzheimer’s disease is a prevalent and debilitating neurodegenerative condition that profoundly affects a patient’s daily functioning with progressive cognitive decline,which can be partly attributed to impaired hippocampal neurogenesis.Neurogenesis in the hippocampal dentate gyrus is likely to persist throughout life but declines with aging,especially in Alzheimer’s disease.Recent evidence indicated that RNA-binding protein 8A(Rbm8a)promotes the proliferation of neural progenitor cells,with lower expression levels observed in Alzheimer’s disease patients compared with healthy people.This study investigated the hypothesis that Rbm8a overexpression may enhance neurogenesis by promoting the proliferation of neural progenitor cells to improve memory impairment in Alzheimer’s disease.Therefore,Rbm8a overexpression was induced in the dentate gyrus of 5×FAD mice to validate this hypothesis.Elevated Rbm8a levels in the dentate gyrus triggered neurogenesis and abated pathological phenotypes(such as plaque formation,gliosis reaction,and dystrophic neurites),leading to ameliorated memory performance in 5×FAD mice.RNA sequencing data further substantiated these findings,showing the enrichment of differentially expressed genes involved in biological processes including neurogenesis,cell proliferation,and amyloid protein formation.In conclusion,overexpressing Rbm8a in the dentate gyrus of 5×FAD mouse brains improved cognitive function by ameliorating amyloid-beta-associated pathological phenotypes and enhancing neurogenesis.

复方莪术散对子宫内膜异位症患者cAMP-PKA信号通路及MMP-2和TIMP-2表达的影响

目的:观察复方莪术散对子宫内膜异位症患者c AMP-PKA信号通路及异位内膜基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)和基质金属蛋白酶组织抑制剂(tissue inhibitor of metalloproteinase-2,TIMP-2)表达的影响。方法:86例子宫内膜异位症患者随机分为对照组和观察组各43例。对照组给予孕三烯酮胶囊和手术治疗;观察组在对照组治疗基础上给予复方莪术散。两组均治疗3个月。比较两组患者治疗前后血清肿瘤标志物糖抗原(carbohydrate antigen-125,CA125)、CA199、异位内膜组织和在位内膜组织cyclin D、P16、MMP-2、MMP-2 mRNA、TIMP-2和TIMP-2 mRNA水平。结果:与治疗前比较,治疗后两组患者的血清CA125、CA199水平均降低,差异有统计学意义(P<0.05);与对照组比较,观察组患者治疗后血清CA125、CA199水平低于对照组(P<0.05);观察组异位内膜组织中cyclin D、MMP-2 mRNA、MMP-2蛋白表达水平明显低于对照组(P<0.05);观察组异位内膜P16、TIMP-2 mRNA、TIMP-2蛋白表达水平明显高于对照组(P<0.05)。结论:复方莪术散可以通过调节c AMP-PKA信号通路,提高子宫内膜异位症患者异位内膜TIMP-2 mRNA、TIMP-2蛋白表达水平,降低MMP-2 mRNA、MMP-2蛋白表达水平。