Effects of TNF-α-mediated insulin resistance on INSIG2 and SCAP expressions in vivo

AIM: To investigate the effects of TNF-α induced insulin resistance (IR) on INSIG1, INSIG2, SCAP and SREBP expressions in mice. METHODS: Male C57BL/6J mice were randomly divided into 4 groups. The mice were given an intraperitoneal injection of TNF-α (6 μg·kg-1·d-1; 3 μg·kg-1·d-1 and 1 μg·kg-1·d-1) and saline (NC group) twice daily for 7 d. The insulin sensitivity and glucose metabolism in awaken mice were evaluated by intravenous glucose tolerance test (IVGTT). The mRNA expression and protein levels of gene were measured by RT-PCR and Western blotting. RESULTS: After TNF-α treatment, fasting blood glucose (FBG), plasma insulin and free fatty acids (FFA) were significantly elevated in TNF-α (6 μg·kg-1·d-1) group compared to NC, TNF-α (1 μg·kg-1·d-1) and TNF-α (3 μg·kg-1·d-1) groups (P<0.01 and P<0.05, respectively). There was a lower glucose tolerance in TNF-α (6 μg·kg-1·d-1) group than that in other three groups during IVGTT. In TNF-α (6 μg·kg-1·d-1) group, the insulin release of glucose-stimulation was higher than that in NC and TNF-α (1 μg·kg-1·d-1) groups (P<0.01 and P<0.05). The INSIG2 mRNA expression of adipose tissues in TNF-α (6 μg·kg-1·d-1) group was significantly increased compared with NC group (P<0.01), and INSIG2 protein levels were also increased (P<0.05). In TNF-α treatment mice, SCAP mRNA level in adipose tissues was significantly up-regulated than that in the controls (P<0.05). The mRNA expressions of INSIG1 and SREBP1 in two groups were not significantly changed (P>0.05). CONCLUSION: In TNF-α induced insulin resistance, INSIG2 and SCAP may be involved in the pathways of lipid metabolism.     

C/EBPα is involved in transcriptional regulation of mVGLUT2 gene expression

AIM: The main purpose of this study is to investigate the regulatory role of C/EBPα in mouse vesicular glutamate transporter 2(mVGLUT2) gene expression. METHODS: The promoter region of mVGLUT2 was cloned to PGL3-basic vector. Site-direction mutation was used to identify the CCAAT-enhancer-binding protein α(C/EBPα) binding site. The promoter activity was observed by luciferase system. The binding between C/EBPα protein and mVGLTU2 promoter region was determined by EMSA. The C/EBPα gene expression was inhibited by its specific siRNA. RESULTS: mVGLUT2 promoter activity decreased about 50% after mutation of C/EBPα binding site. EMSA showed that C/EBPα protein bound onto mVGLUT2 promoter region. Meanwhile, when C/EBPα gene expression was inhibited by its specific siRNA, mVGLUT2 promoter activity, mRNA level and protein level were decreased about 60%, 40% and 45%, respectively. CONCLUSION: C/EBPα is involved in the regulation of mVGLUT2 gene expression.     

Effect of inhibiting nuclear factor-kappa B on TNF-α-induced expression of angiotensinogen and production of angiotensinⅡ in glomerular mesangial cells

AIM: To study the effect of inhibiting nuclear factor-kappa B (NF-κB) activity on the expression of angiotensinogen (AGT) and the production of angiotensinⅡ (AngⅡ) induced by tumor necrosis factor-α (TNF-α) in glomerular mesangial cells (MCs) of SD rats. METHODS: The MCs of SD rats were isolated and divided into three groups as follows: control; MCs treated with TNF-α, and the MCs treated with TNF-α + pyrrolidinedithiocarbamate (PDTC). The activity of nuclear factor-kappa B was measured by electrophoretic mobility shift assay. The expression of AGT was determined by RT-PCR for mRNA and Western blotting for protein. The concentration of angiotensinⅡ in supernatant was measured by RIA. RESULTS: The NF-κB activity in the MCs treated with TNF-α (20.67±9.14)×102 μg/cell was significantly higher than that in control cells ［(8.25±4.35)×102 μg/cell, P<0.01］ and the MCs treated with TNF-α+PDTC ［(7.20±4.57)×102 μg/cell, P<0.01］, and no significant difference was found between control and the MCs treated with TNF-α＋PDTC (P>0.05). The AGT mRNA level in the MCs treated with TNF-α (0.27±0.05) was higher than that in the control cells (0.20±0.05, P<0.05), and no significant difference was observed when compared with that in the MCs treated with TNF-α＋PDTC (0.22±0.06, P>0.05). The expression of AGT protein in the MCs treated with TNF-α (0.60±0.19) μg/cell was higher than that in the control ［(0.37±0.15)μg/cell, P＜0.05］ and the MCs treated with TNF-α＋PDTC ［(0.37±0.17)μg/cell, P<0.05］, and no significance was found between the MCs treated TNF-α＋PDTC and the control (P>0.05). The AngⅡ level in supernatant of cultured MCs treated with TNF-α ［(9.73±2.38)×10-5 ng·L-1/cell］ was significantly higher than that in the control ［(7.50±1.51)×10-5 ng·L-1/cell, P<0.05］ and in the MCs treated with TNF-α＋PDTC ［(6.94±1.46)×10-5 ng·L-1/cell, P<0.05］, however, the difference between the MCs treated with TNF-α＋PDTC and the control was of no significance (P>0.05). CONCLUSION: TNF-α activates the NF-κB in glomerular MCs, induces the AGT expression and the production of AngⅡ. Inhibition of NF-κB decreases the AGT expression and the production of AngⅡ. Therefore, the effects of TNF-α on AGT and AngⅡ may be mediated by NF-κB.     

Reversal of multidrug resistance by transfection of tumor necrosis factor α and MDR1 antisense RNA into multidrug resistant breast cancer cell line

AIM: To study the reversal effects of multidrug resistance by transfecting tumor necrosis factor α (TNF-α) cDNA and multidrug resistant 1 (MDR1) gene antisense RNA into multidrug resistant breast cancer cell line MCF-7/ADR. METHODS: The recombinant vector of enhanced green fluorescent protein (EGFP) with MDR1 antisense RNA and recombinant vector of red fluorescent protein (DsRed2) with TNF-α cDNA were constructed by RT-PCR and DNA recombinant techniques. The recombinant vectors were transfected into multidrug resistant breast cancer cell line MCF-7/ADR. The cell growth curves, cell apoptosis rates, MDR1 gene expression at mRNA and P-gp levels, and the sensitivity to ADR were determined before and after the transfection. RESULTS: After the transfection, cells showed lower growth rate, higher apoptosis rate, lower MDR1 expression at mRNA and P-gp levels, and the sensitivity to ADR increased significantly. CONCLUSION: Transfection of TNF-α cDNA and MDR1 antisense RNA into multidrug resistant breast cancer cells may have good effects on reversal of multidrug resistance.     

Effects of thrombospondin 1 on rat cardiac fibroblasts induced by transforming growth factor β1

AIM:To investigate the effects of thrombospondin 1 on transforming growth factor β₁ induced rat cardiac fibroblasts (CFs). METHODS: CFs of neonatal Sprague -Dawley (SD) rats were isolated with the method of digestion and differential anchoring velocity. The proliferation and collagen synthesis of rat CFs were observed with MTT and hydroxyproline. The expression of TSP-1mRNA was analyzed by RT-PCR.RESULTS: The dose and time-dependent effects of TGF-β₁ were observed. Expression of TSP-1 was increased significantly (P<0.01). Stimulation of CFs with TGF-β₁ (20 μg/L, 24 h) significantly increased CFs proliferation and collagen synthesis (P<0.01). TSP-1 antisense oligonucleotide effectively inhibited TGF-β₁ induced CFs proliferation and collagen synthesis (P<0.01).CONCLUSION:The proliferation and collagen synthesis of CFs induced by TGF-β₁ are inhibited by TSP-1 antisense oligonucleotide, which may exert helpful effect on anti-fibrosis.     

Effects of zanthoxylum seed oilA2 on NF-кB signaling pathway and inflammatory factor in lung tissue of asthmatic mice

AIM: To investigate the dynamic influence of zanthoxylum seed oilA2 (ZSOA2) on NF-кB signaling pathway and inflammatory factor in pulmonary tissue of asthmatic mice. METHODS: The suspensoid (0.2 mL containing 20% albumin hydroxide and 10% ovalbumin) was administered by intraperitoneal injection to sensitize the BALB/c mice on day 1, then 0.4% ovalbumin solution (50 μL in phosphate buffer fluid) was dripped into the respiratory tract through nasal cavity to establish the asthmatic mouse model. After dripped ovalbumin for 24 h, 48 h, 3 d, 7 d and 14 d, the mice were killed at specified time points. The contents of interleukin-4 (IL-4), interleukin-5 (IL-5) and interferon-γ (IFN-γ) in bronchoalveolar lavage fluid (BALF) were determined by ELISA. The pathological changes of the lung tissues were observed with HE staining. The inflammatory cell counts were conducted by Eosin staining. The protein levels of adhesion molecule and the molecules of NF-κB signaling pathway in lung tissues were determined by Western blotting. RESULTS: In ZSOA2 treated mice, the pathological injury of the lung was significantly attenuated as compared to the model mice, the counts of eosinophils and lymphocytes were reduced obviously in lung bronchial area of asthmatic mice at all observed time points (P<0.05). The levels of IL-5 and IL-4 decreased and IFN-γ increased in BALF. The results of Western blotting showed that ZSOA2 down-regulated the protein levels of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, I kappa B kinase alpha-α and phosphorylation inhibitory-κB. ZSOA2 also up-regulated the protein levels of tumor necrosis factor receptor 1 and phosphorylation nuclear factor-kappaB in lung tissue at all observed time points. CONCLUSION: ZSOA2 has therapeutic effect on asthma by down-regulating the protein expression of IκB-β and p-IκB, inhibiting the releases of cytokines and chemotactic factors, and attenuating the infiltration of inflammatory cells in the lungs of ovalbumin challenged asthma mice.     

Effects of curcumin on actinomycin D/TNF-α-induced injury in PC12 cells and rat hippocampal neurons

AIM: To study the possible mechanism of curcumin on actinomycin D (ActD)/tumor necrosis factor α (TNF-α)-induced injury in PC12 cells and rat hippocampal neurons. METHODS: PC12 cells were divided into control group, TNF-α group, ActD group, curcumin group, ActD/TNF-α group and curcumin+ActD/TNF-α group. The cells were cultured for 24 h. Inverted fluorescence microscopy was used to observe the morphological changes of the cells in each group. Annexin V/PI double staining was applied to analyze the apoptosis of PC12 cells. The level of intracellular Ca²⁺ was detected by Fluo-3 AM staining. Rat hippocampal slices were prepared and divided into the same groups as the PC12 cells. Extracellular microelectrode recording technique was used to observe and calculate the changes of long-term potentiation (LTP) in different groups. RESULTS: Apoptosis of PC12 cells was induced by ActD/TNF-α. Curcumin protected the PC12 cells from ActD/TNF-α-induced apoptosis (P<0.05). ActD/TNF-α increased the intracellular Ca²⁺ concentration. Curcumin significantly reduced ActD/TNF-α-induced apoptosis by decreasing the intracellular Ca²⁺ concentration (P<0.05), inversed the effect of ActD/TNF-α on LTP in hippocampal slices, and improved the synaptic plasticity (P<0.05). CONCLUSION: Curcumin protects ActD/TNF-α-induced neuronal damage by depressing the intracellular Ca²⁺ concentration and maintaining the homostasis of intracellular calcium.     

Effects of TNF-α-induced changes of expression and activity of 11-β HSD-1 on the insulin sensitivity in 3T3-L1 adipocytes

AIM: To observe the effects of tumor necrosis factor-α（TNF-α）-induced changes of expression and activity of 11-beta-hydroxysteroid dehydrogenase type1(11-β HSD-1) on the insulin sensitivity in 3T3-L1 adipocytes.METHODS: 3T3-L1 adipocytes were treated with TNF-α and TNF-α combined with aspirin, 2’-hydroxyflavanone or RU486, then mRNA expression and activity of 11-β HSD-1 and insulin-stimulated glucose uptake were examined.RESULTS: TNF-α increased expression and activity of 11-β HSD-1 in 3T3-L1 adipocytes, and decreased insulin-stimulated glucose uptake. Aspirin decreased expression and activity of 11-β HSD-1 induced by TNF-α, and alleviated the inhibiting effect of TNF-α on insulin-stimulated glucose uptake. 11-β HSD-1 specific inhibitor 2’-hydroxyflavanone and cortisol-receptor antagonist RU486 also alleviated the inhibiting effect of TNF-α on insulin-stimulated glucose uptake. CONCLUSION: TNF-α may decrease the insulin sensitivity in 3T3-L1 adipocytes through increasing expression and activity of 11-β HSD-1.     

Detection and differential analysis of fruit organic acids among different local wrinkled papaya varieties(Chaenomeles speciosa ) by GC-MS北大核心CSCD

【Objective】Organic acid plays a key role in affecting fruit flavor by changing acid-sugar ratio. And GC-MS is also an important detecting platform to inspect fruit organic acids due to its stable, sensitive and accurate features. With methyl ester derivatization, the objective organic acids could be effectively detected for their reduction of ingredient polarity. To provide the basic data for Chaenomeles speciosa (Sweet) Nakai fruit quality improvement, the the organic acid inspection of the fruit sampleswas carried out from 10 main producing areas in China and their compositions and contentswere disclosed by extracting with methal, derivating with methyl esterification and detecting by means of GC-MS.【Methods】In this study, 10 local varieties were used as materials that were collected from 10 main producing areas in China. After methanol extraction and methyl-ester derivatization, the compositions and contents of organic acids with each sample were comprehensively determined by GC- MS. The derivatives were analyzed by a Shimadzu GC-MS 2010 Qplus with a Rxt-5MS weak polar capillary MS column(30 m × 0.25 mm, 0.25 μm, Shimadzu Technology). Helium was used as the carrier gas at 0.87 mL · min- 1 with a split ratio of 53:1 for the testing solution. The GC-MS detecting time was 35 min. Qualitative retrieval was conducted with similarity searching in NIST08 and NIST08S coupled with Kovats Reservation Index(RI value) matching and quantitative analysis was performed by an external standard method and the ingredient peak responding value was adjusted according to the n-alkanes mixed standards that came from USA O2Si calibration standards company. The difference in objective organic acids of all the 10 local variety samples was done through the Excel 2007 software and the Hierarchical Cluster Analysis(HCA) of organic acid composition among different samples was completed with SPSS20.0.【Results】Six standards for organic acids including malic acid, citric acid, oxalic acid, succinic acid, fumaric acid and oleic acid as well as their gradient regression equations showed that there was a high correlation between the standard concentration and their component peak area(R2 ≥ 92.9%). All baselines of the TICs were stable and all the component peaks were evenly distributed during the detecting period and their resolution was high. So the method for extracting by methanol, derivating by methyl ester and detecting by GC-MS was stable and reliable. A total of 43 organic acids including 9 short-chain carboxylic acids, 22 long-chain fatty acids, 4 aromatic dicarboxylic acids, 4 mono-basic phenol acids and 2 amino acids were identified from the 10 fruit samples of different producing areas in China. The top 10 organic acids with the highest contents were dl-malic acid, citric acid, hexadecanoic acid, 9,12-octadecadienoic acid, 9-octadecenoic acid, (+/-)-10-hydroxy-octadecanoic acid, levulinic acid, stearic acid, 9,10-dihydroxy-octadecanoic acid and benzoic acid, respectively. There were 33 common ingredient peaks among the total 10 local varieties and their total contents of organic acids were between 85.02-170.76 mg·g-1. From high to low content, it showed like this: Linyi of Shandong > Jinghong of Yunnan > Zheng’an of Guizhou > Chun’an of Zhejiang > Qijiang of Chongqing> Changyang of Hubei > Xuancheng of Anhui > Yilong of Sichuan > Nanning of Guangxi > Baihe of Shaanxi. The total organic acid content had an extremely significant positive correlation with the long-chain fatty acids, mainly including hexadecanoic acid, 9,12-octadecadienoic acid and 9-octadecenoic acid; a significant positive correlation with the short-chain carboxylic acids, mainly consisting of dl-malic acid and citric acid; a negative correlation with the aromatic carboxylic acids, mainly containing benzoic acid. Analysis of the cluster according to 33 common components showed that all the fruit samples were classified into 4 categories when their clustering distance was 5: Yilong of Sichuan, Xuancheng of An’hui, Baihe of Shaanxi, Nanning of Guangxi, and Changyang of Hubei were clustered into the first group; Qijiang of Chongqing and Chun’an of Zhejiang were clustered into the second branch. Zheng’an of Guizhou and Linyi of Shandong were clustered into the third group, and Jinghong of Yunnan was alone clustered into the fourth group. The Hierarchical Cluster Analysis (HCA) coupled with the total organic acid content showed that Zheng’an of Guizhou and Linyi of Shandong belonged to the high-acid varieties, Qijiang of Chongqing and Chun’an of Zhejiang were the middle-acid varieties, and Yilong of Sichuan, Baihe of Shaanxi and Nanning of Guangxi belonged to the low-acid varieties.【Conclusion】Compared with the acid-base titration method and HPLC, GC-MS method not onlywas more stable, sensitive and accurate, but also could realize a qualitative identification of more chemical components. So it was much better to be used in the determination of total organic acid contents in fruits and their derived products. The results showed that there was a small difference among the different local varieties and an obvious difference existed in organic acid compositions and contents among the samples from different producing areas in China. And dl-malic acid and citric acid were the main components in most of C. speciosa fruit samples from 10 producing areas in China. Therefore, it belonged to the fruit type of malic acid accumulating mode. © 2019 Journal of Fruit Science     

Role of protein tyrosine phosphatase SHP-2 in cardiac fibroblasts proli-feration stimulated by Ang Ⅱ

AIM: To investigate the potential role of Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP-2) in the proliferation of cardiac fibroblasts induced by angiotensin Ⅱ (Ang Ⅱ). METHODS: The neonatal rat cardiac fibroblasts were separated by trypsin digestion. The cardiac fibroblasts were identified by vimentin of immunochemical staining. The proliferation of the cardiac fibroblasts with or without Ang Ⅱ stimulation was measured by MTT assay. Adenoviruses containing SHP-2 gene were transfected into cardiac fibroblasts to overexpress SHP-2. SHP-2 was inhibited by its inhibitor NSC-87877. RESULTS: The proliferation of the cardiac fibroblasts was increased in a dose-dependent manner by the stimulation of Ang Ⅱ and the maximum concentration of Ang II for cell proliferation was 10^-7 mol/L. SHP-2 promoted the proliferation of cardiac fibroblasts under the stimulation of Ang Ⅱ. The proliferation rate in mutant group was higher than that in wild-type group (P<0.01). Inhibition of SHP-2 by NSC-87877 attenuated the proliferation. CONCLUSION: The growth promoting effect of Ang Ⅱ on cardiac fibroblasts is regulated by SHP-2.