Expression, purification, and functional verification of recombinant human glycoprotein hormone beta 5/alpha 2 fusion protein in CHO-S cells / 中国药理学通报

Aim To express and purify recombinant hCGH-CTP fusion protein in high-density suspension culture of Chinese hamster ovary cells (CHO-S), and to verify the lipid accumulation effect of rhCGH-CTP on 3T3-L1 mature adipocytes. Methods The recombinant protein expression vector (pcDNA3. 1-rhCGH-CTP) was constructed, achieved by fusing the human glycoprotein hormone beta 5/alpha 2 cDNA with CTP Linker. The expression plasmid was transiently transfected into the suspended CHO-S to express rhCGH-CTP protein and then purified, and the protein biological activity was verified. Intervention with 3T3-L1 mature adipocyte cells for 24 h was performed to detect the changes of intracellular triglyceride (TG) level. Results Western blot results showed that rhCGH-CTP protein was successfully expressed in CHO-S cells, and the yield was up to 715. 4 mg • L~ . The secreted protein was purified by AKTA pure system with higher purity that was up to 90% as identified by SDS-PAGE. In addition, the intracellular cAMP content of mature adipocytes with high expression of TSHR gene significantly increased after intervention with different concentrations of rhCGH-CTP protein by ELISA kit, indicating that rhCGH-CTP protein had biological activity. Oil red 0 staining showed that compared with the control group, the lipid content of mature adipocytes in the intervention groups with different concentrations of rhCGH-CTP protein significantly decreased (P < 0. 05) . Conclusions The rhCGH-CTP protein has been successfully expressed and purified with biological activity, and effectively reduce TG. This research provides an important theoretical basis for further revealing the physiological role of CGH protein and its potential application in clinical practice.

Impairment of spermatogenesis and sperm motility by the high-fat diet-induced dysbiosis of gut microbes.

OBJECTIVE: High-fat diet (HFD)-induced metabolic disorders can lead to impaired sperm production. We aim to investigate if HFD-induced gut microbiota dysbiosis can functionally influence spermatogenesis and sperm motility. DESIGN: Faecal microbes derived from the HFD-fed or normal diet (ND)-fed male mice were transplanted to the mice maintained on ND. The gut microbes, sperm count and motility were analysed. Human faecal/semen/blood samples were collected to assess microbiota, sperm quality and endotoxin. RESULTS: Transplantation of the HFD gut microbes into the ND-maintained (HFD-FMT) mice resulted in a significant decrease in spermatogenesis and sperm motility, whereas similar transplantation with the microbes from the ND-fed mice failed to do so. Analysis of the microbiota showed a profound increase in genus Bacteroides and Prevotella, both of which likely contributed to the metabolic endotoxaemia in the HFD-FMT mice. Interestingly, the gut microbes from clinical subjects revealed a strong negative correlation between the abundance of Bacteroides-Prevotella and sperm motility, and a positive correlation between blood endotoxin and Bacteroides abundance. Transplantation with HFD microbes also led to intestinal infiltration of T cells and macrophages as well as a significant increase of pro-inflammatory cytokines in the epididymis, suggesting that epididymal inflammation have likely contributed to the impairment of sperm motility. RNA-sequencing revealed significant reduction in the expression of those genes involved in gamete meiosis and testicular mitochondrial functions in the HFD-FMT mice. CONCLUSION: We revealed an intimate linkage between HFD-induced microbiota dysbiosis and defect in spermatogenesis with elevated endotoxin, dysregulation of testicular gene expression and localised epididymal inflammation as the potential causes. TRIAL REGISTRATION NUMBER: NCT03634644.

Protective effects of ginsenoside Rg1 against hydrogen peroxide-induced injury in human neuroblastoma cells.

The active ingredient of ginseng, ginsenosides Rg1, has been shown to scavenge free radicals and improve antioxidant capacity. This study hypothesized that ginsenosides Rg1 has a protective role in human neuroblastoma cells injured by H2O2. Ginsenosides Rg1 at different concentrations (50 and 100 µM) was used to treat H2O2 (150 µM)-injured SH-SY5Y cells. Results demonstrated that ginsenoside Rg1 elevated the survival rate of SH-SY5Y cells injured by H2O2, diminished the amount of leaked lactate dehydrogenase, and increased superoxide dismutase activity. Ginsenoside Rg1 effectively suppressed caspase-3 immunoreactivity, and contributed to heat shock protein 70 gene expression, in a dose-dependent manner. These results indicate that ginsenoside Rg1 has protective effects on SH-SY5Y cells injured by H2O2 and that its mechanism of action is associated with anti-oxidation and the inhibition of apoptosis.

Role of synaptic activity in the regulation of amyloid beta levels in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia. Accumulation of amyloid-beta (Aß) peptides is regarded as the critical component associated with AD pathogenesis, which is derived from the amyloid precursor protein (APP) cleavage. Recent studies suggest that synaptic activity is one of the most important factors that regulate Aß levels. It has been found that synaptic activity facilitates APP internalization and influences APP cleavage. Glutamatergic, cholinergic, serotonergic, leptin, adrenergic, orexin, and gamma-amino butyric acid receptors, as well as the activity-regulated cytoskeleton-associated protein (Arc) are all involved in these processes. The present review summarizes the evidence for synaptic activity-modulated Aß levels and the mechanisms underlying this regulation. Interestingly, the immediate early gene product Arc may also be the downstream signaling molecule of several receptors in the synaptic activity-modulated Aß levels. Elucidating how Aß levels are regulated by synaptic activity may provide new insights in both the understanding of the pathogenesis of AD and in the development of therapies to slow down the progression of AD.

Aß-induced microglial cell activation is inhibited by baicalin through the JAK2/STAT3 signaling pathway.

Baicalin has shown multiple neuroprotective biological activities, including antiapoptotic and anti-inflammatory functions in neurodegeneration diseases. However, whether baicalin can regulate Aß-induced microglial activation or inhibit inflammatory cytokine secretion has not been confirmed. We demonstrated that baicalin can inhibit beta amyloid peptides (Aß42)-induced BV2 microglial cell proliferation, reduce the expression of CD11b, decrease chemotactic ability of BV2 cells and significantly inhibit the secretion of IL-6, TNF-α and NO. Moreover, baicalin pretreatment can effectively inhibit Aß-induced phosphorylation of JAK2 and STAT3. Baicalin can inhibit Aß-induced microglial cell activation by regulating the JAK2/STAT3 signaling pathway in AD transgenic mice. The modulation of microglial proliferation, activation and secretion by baicalin could be a promising therapeutic option for the treatment of Alzheimer's disease.

RXRα inhibits the NRF2-ARE signaling pathway through a direct interaction with the Neh7 domain of NRF2.

The transcription factor NRF2 (NFE2L2) is a pivotal activator of genes encoding cytoprotective and detoxifying enzymes that limit the action of cytotoxic therapies in cancer. NRF2 acts by binding antioxidant response elements (ARE) in its target genes, but there is relatively limited knowledge about how it is negatively controlled. Here, we report that retinoic X receptor alpha (RXRα) is a hitherto unrecognized repressor of NRF2. RNAi-mediated knockdown of RXRα increased basal ARE-driven gene expression and induction of ARE-driven genes by the NRF2 activator tert-butylhydroquinone (tBHQ). Conversely, overexpression of RXRα decreased ARE-driven gene expression. Biochemical investigations showed that RXRα interacts physically with NRF2 in cancer cells and in murine small intestine and liver tissues. Furthermore, RXRα bound to ARE sequences in the promoters of NRF2-regulated genes. RXRα loading onto AREs was concomitant with the presence of NRF2, supporting the hypothesis that a direct interaction between the two proteins on gene promoters accounts for the antagonism of ARE-driven gene expression. Mutation analyses revealed that interaction between the two transcription factors involves the DNA-binding domain of RXRα and a region comprising amino acids 209-316 in human NRF2 that had not been defined functionally, but that we now designate as the NRF2-ECH homology (Neh) 7 domain. In non-small cell lung cancer cells where NRF2 levels are elevated, RXRα expression downregulated NRF2 and sensitized cells to the cytotoxic effects of therapeutic drugs. In summary, our findings show that RXRα diminishes cytoprotection by NRF2 by binding directly to the newly defined Neh7 domain in NRF2.

Anti-tumor effect of rutin on human neuroblastoma cell lines through inducing G2/M cell cycle arrest and promoting apoptosis.

AIMS: To further investigate the antineuroblastoma effect of rutin which is a type of flavonoid. METHODS: The antiproliferation of rutin in human neuroblastoma cells LAN-5 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Chemotaxis of LAN-5 cells was assessed using transwell migration chambers and scratch wound migration assay. The cell cycle arrest and apoptosis in a dose-dependent manner was measured by flow cytometric and fluorescent microscopy analyses. The apoptosis-related proteins BAX and BCL2 as well as MYCN mRNA express were determined by RT-PCR analysis. Secreted TNF- α level were determined using specific enzyme-linked immunosorbent assay kits. RESULTS: Rutin significantly inhibited the growth of LAN-5 cells and chemotactic ability. Flow cytometric analysis revealed that rutin induced G2/M arrest in the cell cycle progression and induced cell apoptosis. The RT-PCR showed that rutin could decrease BCL2 expression and BCL2/BAX ratio. In the meantime, the MYCN mRNA level and the secretion of TNF- α were inhibited. CONCLUSION: These results suggest that rutin produces obvious antineuroblastoma effects via induced G2/M arrest in the cell cycle progression and induced cell apoptosis as well as regulating the expression of gene related to apoptosis and so on. It supports the viability of developing rutin as a novel therapeutic prodrug for neuroblastoma treatment, as well as providing a new path on anticancer effect of Chinese traditional drug.

Shuyusan-containing serum protects SH-SY5Y cells against corticosterone-induced impairment.

The Chinese herb Shuyusan, whose main constituent is jasminoidin, has been shown to protect SH-SY5Y cells against corticosterone-induced damage. SH-SY5Y cells injured by 400 µmol/L corticosterone were treated with 5 and 30 µg/mL Shuyusan-containing serum. Results revealed that Shuyusan-containing serum elevated the survival rate of SH-SY5Y cells, reduced Bax expression, increased Bcl-2 expression, markedly elevated brain-derived neurotrophic factor mRNA expression, and blocked cell apoptosis. Moreover, the effect of high-dose (30 µg/mL) Shuyusan-containing serum was more remarkable. Therefore, Shuyusan-containing serum appears to protect SH-SY5Y cells against corticosterone-induced impairment by adjusting the expression of apoptosis-associated proteins and brain-derived neurotrophic factor. Moreover, high-dose Shuyusan-containing serum has a protective effect on high-dose corticosterone-induced impairment.

[Effect of intensive rosuvastatin therapy on adhesion molecules and the upstream mechanism in patients with peripheral atherosclerosis].

OBJECTIVE: To investigate the effect of intensive rosuvastatin therapy on adhesion molecules in patients with peripheral atherosclerosis and explore the possible upstream mechanism. METHODS: Twenty asymptomatic patients with peripheral atherosclerosis were enrolled and given 5-20 mg/day rosuvastatin for 3 months. Before and after the treatment, the lipid profile and plasma vascular cell adhesion molecule-1 (VCAM-1) levels were examined. The expression of intercellular adhesion molecule-1 (ICAM-1) in the mononuclear cells was measured using flow cytometry, and the mRNA and protein expressions of peroxisome proliferator-activated receptor γ (PPARγ) were detected using RT-PCR and Western blotting, respectively. RESULTS: Compared with the baseline levels, ICAM-1 expression decreased and PPARγ protein expression increased in the lymphocytes. Rosuvastatin therapy did not produce obvious effects on plasma VCAM-1 level or ICAM-1 expression in the monocytes in these patients. CONCLUSION: Rosuvastatin produces anti-inflammatory effects by decreasing the expression of ICAM-1 in mononuclear cells, and its upstream mechanism may involve the PPARγ pathway.

Antidepressant-like effects of shuyusan in rats exposed to chronic stress: effects on hypothalamic-pituitary-adrenal function.

This study was to investigate antidepressant activities of Shuyusan (a Chinese herb), using a rats model of depression induced by unpredictable chronic mild stress (UCMS). The administration groups were treated with Shuyusan decoction for 3 weeks and compared with fluoxetine treatment. In order to understand the potential antidepressant-like activities of Shuyusan, tail suspension test (TST) and forced swimming test (FST) were used as behavioral despair study. The level of corticotropin-releasing factor (CRH), adrenocorticotropic hormone (ACTH), corticosterone (CORT) and hippocampus glucocorticoid receptor expression were examined. After modeling, there was a significant prolongation of immobility time in administration groups with the TST and FST. High-dose Shuyusan could reduce the immobility time measured with the TST and FST. The immobility time in high-dose herbs group and fluoxetine group was increased significantly compared with the model group. After 3 weeks herbs fed, the serum contents level of CRH, ACTH, and CORT in high-dose herb group was significantly decreased compared to the model group. The result indicated that Shuyusan had antidepressant activity effects on UCMS model rats. The potential antidepressant effect may be related to decreasing glucocorticoid levels activity, regulating the function of HPA axis, and inhibiting glucocorticoid receptor expression in hippocampus.