Baicalin attenuates pancreatic fibrosis by inhibiting TGF-β1/TAK-NF-κB signaling pathway in chronic pancreatitis mice

AIM: To explore the role of transforming growth factor-β1 (TGF-β1)/TGF-β-activated kinase (TAK)-nuclear factor-κB (NF-κB) signaling pathway in chronic pancreatitis (CP) mice and the effect of baicalin on pancreatic fibrosis in the mice. METHODS: Kunming mice (n=58) were randomly divided into 3 groups, including control group, CP group and baicalin group. The mice in CP group and baicalin group were intraperitoneally injected with 20% L-arginine. After 2 weeks of CP, the mice in baicalin group were intraperitoneally injected with baicalin (100 mg/kg, once a day). At 2 weeks, 4 weeks and 6 weeks after modeling, the mice were anesthetized and sacrificed. The morphological changes of the pancreas were observed by HE and Masson staining. The serum level of TGF-β1 was analyzed by ELISA. The expression of fibronectin (FN) and NF-κB in the pancreas was observed by immunohistochemistry staining. The protein levels of transforming growth factor-β receptor type Ⅰ (TGF-βRⅠ), phosphorylated TAK1 (p-TAK1) and NF-κB in the pancreas were determined by Western blot. The mRNA expression of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloprotease-1 (TIMP-1) was detected by real-time PCR. RESULTS: At 2 weeks, 4 weeks and 6 weeks after intraperitoneal injection of L-arginine, the pancreatic tissues were obviously injured and exhibited different degrees of fibrosis, and FN expression was significantly increased. After treatment with baicalin, the degrees of pancreatic injury and fibrosis were significantly attenuated and the expression of FN was reduced (P<0.01). Compared with control group, the protein levels of TGF-β1, TGF-βRⅠ, p-TAK1, NF-κB and TIMP-1 in the pancreas of CP group were significantly increased, and the expression of MMP-1 was decreased at each time point. In baicalin group, the protein levels of TGF-β1, TGF-βRⅠ, p-TAK1, NF-κB and TIMP-1 were significantly decreased, and the expression of MMP-1 was markedly increased at the corresponding time points compared with CP group (P<0.01). CONCLUSION: Baicalin effectively atte-nuates pancreatic fibrosis by inhibiting the activation of TGF-β1/TAK-NF-κB signaling pathway and regulating the balance of MMP-1/TIMP-1.     

β2m基因在猪肾细胞系PK-15中的表达研究

为研究猪肾细胞系PK-15中Beta 2微球蛋白（Beta 2 microglobulin，β2m）基因的表达情况，提取细胞总RNA，经RT-PCR扩增β2m。回收后的基因克隆至pMD18-T载体，获得重组质粒，经EcoR Ⅰ和Hind Ⅲ双酶切筛选后，阳性克隆送生物公司测序。序列经GENETYX version 9.0编辑分析，通过DNAMAN version 5.2.2与其他猪β2m基因进行序列比对分析，利用Mega 5.0的NJ法进一步分析其与其他物种β2m的分子进化关系，并在此基础上利用SWISS-MODEL和PDBsum预测该基因编码的成熟肽二级结构和三级结构。提取PK-15总蛋白，Western blotting检测和分析细胞中β2m的表达。结果显示，经RT-PCR扩增，成功获得β2m条带，大小约360 bp。经测序发现PK-15-β2m基因为364 bp，共编码118个氨基酸，其中成熟肽为98个氨基酸，N端信号肽含有20个氨基酸。序列分析证实PK-15细胞中β2m基因未发生突变；分子进化分析显示，PK-15-β2m与牛的亲缘关系最近，其次为羊、马等。多重序列比对发现，PK-15-β2m与牛、羊、马β2m成熟肽均为98个氨基酸，而其他物种β2m成熟肽为99个氨基酸；二级结构预测显示，PK-15-β2m成熟肽主要以β-折叠、β-转角和γ-转角为主，不含有α-螺旋。三级结构预测显示，PK-15-β2m成熟肽主要由7条β-折叠条带构成。Western blotting分析显示β2m在细胞中成功表达。本研究证实了猪肾细胞系PK-15中β2m在核酸和蛋白质水平均稳定表达，为下一步研究PK-15细胞的抗原递呈功能奠定了基础。     

circRNA-42398 inhibits activation of hepatic stellate cells via TGF-?β1/Smads signaling pathway modulation

AIM To study the effect of mouse circular RNA-42398 (mmu_circ_42398) over-expression on the activation of hepatic stellate cells. METHODS Mouse hepatic stellate JS1 cells were cultured and randomly divided into control group, vector group and mmu_circ_42398 over-expression group.mmu_circ_42398 over-expression plasmid vector was constructed, and then transiently transfected into JS1 cells using Lipofectamine 2000. The cells were collected 48 h after transfection. Expression of mmu_circ_42398 was detected by RT-qPCR.The backsplice site of PCR products was verified by sequencing. The protein levels of α-smooth muscle actin (α-SMA), collagen type I （Col I), transforming growth factor β1(TGF-β1), Smad2, Smad3, p-Smad2 and p-Smad3 in the cells were determined by Western blot. RESULTS RT-qPCR results showed that the expression of mmu_circ_42398 was significantly increased after mmu_circ_42398 over-expression vector was transiently transfected into the JS1 cells (P<0.01). The protein expression levels of α-SMA and Col I were significantly decreased(P<0.01), and the phosphorylation levels of Smad2 and Smad3 were decreased significantly in mmu_circ_42398 over expression group (P<0.01). However, the protein expression levels of TGF-β1, Smad2 and Smad3 had no significant change (P>0.05). CONCLUSION mmu-circ-42398 inhibits the activation of hepatic stellate cells via TGF-β1/Smads signaling pathway modulation.     

Adiponectin inhibits decrease in autophagy of rat H9c2 cardiomyocytes induced by β1-adrenergic receptor autoantibodies

AIMTo investigate whether adiponectin inhibits the decrease in autophagy of rat H9c2 cardiomy?ocytes induced by β₁-adrenergic receptor (β₁-AR) autoantibodies (β₁-AA), and to explore its mechanism. METH?ODS: SD rats were actively immunized with β₁-AR extracellular second loop (β₁-AR-ECII) antigen peptide. Affinity chromatography was used to purify β₁-AA in serum of the SD rats. The viability of H9c2 cells was measured by CCK-8 assay. The mRNA levels of LC3B and beclin-1 in the H9c2 cells were detected by real-time PCR. The protein levels of LC3-II, P62, AMP-activated protein kinase (AMPK) and phosphorylated AMPK (p-AMPK) were determined by Western blot. RESULTSPretreatment with adiponectin at 10 μg/L for 1 h reversed the decreased viability of H9c2 cells induced by β₁-AA. Compared with control group, β₁-AA decreased the mRNA expression of LC3B and beclin-1, decreased the protein level of LC3-II, and increased the expression of P62 protein in the H9c2 cells, suggesting that β₁-AA decreased the autophagic flux in cardiomyocytes. Adiponectin obviously reversed β₁-AA-induced decline in autophagic flux, and up-regulated the phosphorylation level of AMPK decreased by β₁-AA. Treatment with AMPK inhibitor Compound C for 30 min, we observed that the mRNA expression of LC3B and beclin-1 and the protein level of LC3-II in the H9c2 cells decreased by β₁-AA were not effectively reversed by adiponectin, but the increase in P62 protein expression was still effectively reversed, indicating that adiponectin increased autophagosome production dependent on the AMPK pathway, but increased autophagosome clearance independent on the AMPK pathway. CONCLUSION Adiponectin inhibits the decreased autophagy of H9c2 cardiomyocytes induced by β₁-AA.     

Effects of Coriaria sinica Maxim’s extract on burn wound healing and scar hyperplasia in rats based on ILK signaling pathway

AIM: To explore the mechanism of Coriaria sinica Maxim’s extract (CSME) promoting burn wound healing in the early stage and inhibiting excessive scar hyperplasia in the later stage, based on the signaling pathways, such as transforming growth factor-β₁ (TGF-β₁) regulated by integrin-linked kinase (ILK) and ILK regulated by PI3K/AKT. METHODS: Female SD rats (n=180; 180~200 g) were randomly divided into 6 groups: normal control (NC) group, vaseline (VL) group, silver sulfadiazine (SS) group and low-, medium- and high-dose of CSME (CSME-L, CSME-M and CSME-H) groups, with 30 rats in each group. Except for the rats in NC group, VL, SS, and 3 doses of CSME were applied to the wound surface of the rats in the corresponding groups every day after II° burn model was made on their waist-back in the condition of chloral hydrate anesthesia. To calculate the healing rate (HR), 10 rats in each experimental group were randomly selected to remove their wound skin for observing the pathologic change, detecting the expression of related proteins by Western blot and RT-qPCR, and checking the collagen shrinkage (SK) by fibroblast culture at the 7th, 14th, and 21st days. RESULTS: The expression of ILK, fibronectin (FN), TGF-β₁, α-smooth muscle actin (α-SMA) and integrin-β₁ (ITG-β₁) at protein and mRNA levels in wound skin of CSME groups was stronger than that in VL group and SS group at the 7th day in a dose-dependent manner, but weaker than that in VL group and SS group at the 21st day (P<0.05). Meanwhile, the protein and mRNA expression of collagen type I (Col I) in CSME groups was stronger than that in VL group and SS group from the 7th day to the 14th day, but weaker than that in VL group and SS group at the 21st day in a dose-dependent manner (P<0.05). However, the protein and mRNA expression of collagen type III (Col III) in CSME groups was weaker than that in VL group and SS group from the 7th day to the 14th day, but stronger than that in VL group and SS group at the 21st day in a dose-dependent manner (P<0.05). The SK of fibroblasts in VL group and SS group was increased continuously over time and reached its peak at 96 h. SK in CSME groups was only higher than that in VL group and SS group at 24 h and 48 h in a dose-dependent manner, but lower than that in VL group and SS group at 96 h (P<0.05). CONCLUSION: CSME promotes burn wound healing in the early stages and inhibits the scar hyperplasia in the later stages. The mechanisms may be related to its multicomponents or multiple-targets to intervene in the signaling pathways such as TGF-β₁ regulated by ILK and ILK regulated by PI3K/AKT. It may also be related to the ratio of Col I and Col III expression.     

饲料β-胡萝卜素水平对镇海林蛙蝌蚪生长发育的影响

为探讨饲料中添加不同剂量的β-胡萝卜素对于镇海林蛙(Rana zhenhaiensis)蝌蚪生长、幼蛙形态和运动表现及肝脏酶活的影响。将镇海林蛙蝌蚪(Gosner 25~26)288尾随机分为4个处理组。各组分别添加0、50、500和1200 mg/kgβ-胡萝卜素,直至镇海林蛙蝌蚪变态发育完全。结果显示:(1)不同处理组间蝌蚪发育中期的体重、体长和尾长组间差异不显著,而蝌蚪中期发育的历期在组间差异显著,对照组中期发育的历期显著快于β-胡萝卜素处理组。(2)不同处理组间蝌蚪的增重率、特定生长率和变态率差异不显著,对照组的变态时间显著短于β-胡萝卜素处理组,β-胡萝卜素处理组则随着浓度的增加而变态时间缩短。(3)不同处理组间幼蛙体长、头长、前臂及手长和后肢长差异显著,但幼蛙体重差异不显著。(4)不同处理组间幼蛙平均跳跃距离差异显著,50和500 mg/kgβ-胡萝卜素处理组幼蛙平均跳跃距离显著大于另外两组。(5)幼蛙的过氧化氢酶(CAT)活力在不同处理组间差异显著,50 mg/kgβ-胡萝卜素组的酶活显著大于其它处理组,但是幼蛙肝脏总超氧化物歧化酶(T-SOD)活力差异不显著。综上所述,饲料中添加500 mg/kgβ-胡萝卜素试验组,可以显著减缓镇海林蛙蝌蚪的早期发育,延长变态时间,形成较大的幼蛙个体,积累能量较多,运动性能显著增强,个体的适合度增加。     

β–1,3葡聚糖酶基因转化提高苹果对斑点落叶病的抗性

为提高苹果抗病性,利用根癌农杆菌介导的叶片转化法,将带有NPTⅡ筛选基因、GUS标记基因及β–1,3葡聚糖酶目的基因质粒导入‘富士’和‘嘎拉’苹果。具有卡那霉素(Kan)抗性的转化株系,经GUS组织化学染色及PCR扩增检测,得到17个‘嘎拉’阳性株系和11个‘富士’阳性株系。选取‘嘎拉’和‘富士’各3个阳性株系进行Southern blot检测,结果表明外源目的基因已整合到所试转化株系中。选取‘嘎拉’及‘富士’各5个阳性株系进行半定量RT-PCR检测,除1个‘富士’株系没有条带外,其余株系均有阳性条带,且各条带的明亮清晰程度有差别,说明不同转基因株系中目的基因的表达量有差异。转化植株离体叶片接种苹果斑点落叶病菌进行抗病性表型检测结果证实,‘嘎拉’10个转化株系中8个株系表现抗病,7个‘富士’转化株系中2个株系表现一定的抗病性,抗病性的增强与目的基因的表达水平有关。     

金黄色葡萄球菌细胞壁变化与β-内酰胺类抗生素耐药的关系

旨在揭示金黄色葡萄球菌针对β-内酰胺类抗生素可能存在细胞壁增厚的耐药机制。2016-2018年间，采集宁夏地区部分奶牛养殖场临床及亚临床型乳腺炎的乳样，通过显色培养基鉴别、镜检及PCR方法，分离鉴定牛乳源金黄色葡萄球菌；利用微量肉汤稀释法测定细菌对14种抗菌药物的耐药性，了解本地区金黄色葡萄球菌的耐药率及多重耐药情况；通过qRT-PCR方法检测细胞壁增厚相关的pbpB、murG、glmU、atlR基因转录丰度，并结合透射电镜进行形态观察，以确定增厚及发生原因。结果显示，分离鉴定出261株牛乳源金黄色葡萄球菌，其中包括9株耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus，MRSA）。药敏试验结果显示，金黄色葡萄球菌对β-内酰胺类抗生素具有较高的耐药率，其中氨苄西林为79.69%，青霉素为78.54%。多重耐药情况是以3、7和8重耐药的菌株居多；其中1株耐药种数达14种之多。qRT-PCR结果表明，4种相关基因的转录丰度均极显著上调（P<0.001或P<0.01）。透射电镜观察发现，甲氧西林敏感的金黄色葡萄球菌（methicillin sensitive Staphylococcus aureus，MSSA）JY21菌株的细胞壁在64和128 μg·mL^-1的青霉素浓度下，较对照组均极显著增厚（P<0.001），并可见细胞壁表面粗糙，有结节状凸起；但药物浓度从64 μg·mL^-1升高至128 μg·mL^-1细胞壁不再显著增厚（P>0.05）。MRSA WLD10菌株细胞壁未出现明显增厚（P>0.05）。综上所述，本地区牛乳源金黄色葡萄球菌针对β-内酰胺类抗生素，存在细胞壁增厚的耐药机制；增厚的原因主要是肽聚糖的过度合成及细胞自溶的减少。与MSSA JY21菌株相比，细胞壁增厚并非MRSA WLD10重要的耐药机制。