The present study was aimed to investigate the role and mechanism of glutaminolysis of cardiac fibroblasts (CFs) in hypertension-induced myocardial fibrosis. C57BL/6J mice were administered with a chronic infusion of angiotensin II (Ang II, 1.6 mg/kg per d) with a micro-osmotic pump to induce myocardial fibrosis. Masson staining was used to evaluate myocardial fibrosis. The mice were intraperitoneally injected with BPTES (12.5 mg/kg), a glutaminase 1 (GLS1)-specific inhibitor, to inhibit glutaminolysis simultaneously. Immunohistochemistry and Western blot were used to detect protein expression levels of GLS1, Collagen I and Collagen III in cardiac tissue. Neonatal Sprague-Dawley (SD) rat CFs were treated with 4 mmol/L glutamine (Gln) or BPTES (5 μmol/L) with or without Ang II (0.4 μmol/L) stimulation. The CFs were also treated with 2 mmol/L α-ketoglutarate (α-KG) under the stimulation of Ang II and BPTES. Wound healing test and CCK-8 were used to detect CFs migration and proliferation respectively. RT-qPCR and Western blot were used to detect mRNA and protein expression levels of GLS1, Collagen I and Collagen III. The results showed that blood pressure, heart weight and myocardial fibrosis were increased in Ang II-treated mice, and GLS1 expression in cardiac tissue was also significantly up-regulated. Gln significantly promoted the proliferation, migration, mRNA and protein expression of GLS1, Collagen I and Collagen III in the CFs with or without Ang II stimulation, whereas BPTES significantly decreased the above indices in the CFs. α-KG supplementation reversed the inhibitory effect of BPTES on the CFs under Ang II stimulation. Furthermore, in vivo intraperitoneal injection of BPTES alleviated cardiac fibrosis of Ang II-treated mice. In conclusion, glutaminolysis plays an important role in the process of cardiac fibrosis induced by Ang II. Targeted inhibition of glutaminolysis may be a new strategy for the treatment of myocardial fibrosis.
Rats ; Mice ; Animals ; Rats, Sprague-Dawley ; Angiotensin II/pharmacology* ; Fibroblasts ; Mice, Inbred C57BL ; Fibrosis ; Collagen/pharmacology* ; Collagen Type I/metabolism* ; RNA, Messenger/metabolism* ; Myocardium/pathology*

This study aims to investigate the role of slient mating-type information regulation 2 homolog 1(SIRT1)/tuberous sclerosis complex 2(TSC2)/mammalian target of rapamycin(mTOR) signaling pathways in the Periplaneta americana extract CⅡ-3-induced senescence of human leukemia K562 cells. K562 cells were cultured in vitro and treated with 0(control), 5, 10, 20, 40, 80, and 160 μg·mL~(-1) of P. americana extract CⅡ-3. Cell counting kit-8(CCK-8) and flow cytometry were employed to examine the proliferation and cell cycle of the K562 cells. Senescence-associated β-galactosidase stain kit(SA-β-gal) was used to detect the positive rate of senescent cells. Mitochondrial membrane potential was detected by flow cytometry. The relative mRNA level of telomerase reverse transcriptase(TERT) was determined by fluorescence quantitative PCR. The mRNA and protein levels of SIRT1, TSC2, and mTOR were determined by fluorescence quantitative PCR and Western blot, respectively. The results showed that CⅡ-3 significantly inhibited the proliferation of K562 cells and the treatment with 80 μg·mL~(-1) CⅡ-3 for 72 h had the highest inhibition rate. Therefore, 80 μg·mL~(-1) CⅡ-3 treatment for 72 h was selected as the standard for subsequent experiments. Compared with the control group, CⅡ-3 increased the proportion of cells arrested in G_0/G_1 phase, decreased the proportion of cells in S phase, increased the positive rate of SA-β-Gal staining, elevated the mitochondrial membrane potential and down-regulated the mRNA expression of TERT. Furthermore, the mRNA expression of SIRT1 and TSC2 was down-regulated, while the mRNA expression of mTOR was up-regulated. The protein expression of SIRT1 and p-TSC2 was down-regulated, while the protein expression of p-mTOR was up-regulated. The results indicated that P. americana extract CⅡ-3 induced the senescence of K562 cells via the SIRT1/mTOR signaling pathway.
Humans ; Animals ; Periplaneta ; Sirtuin 1/genetics* ; K562 Cells ; Signal Transduction ; TOR Serine-Threonine Kinases/genetics* ; RNA, Messenger ; Mammals

This study investigated the effect of Suanzaoren Decoction on the expression of N-methyl-D-aspartate receptors(NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors(AMPAR) in the hippocampus and synaptic plasticity in rats with conditioned fear-induced anxiety. The effect of Suanzaoren Decoction on rat behaviors were evaluated through open field experiment, elevated plus maze experiment, and light/dark box experiment. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of glutamate(Glu) and γ-aminobutyric acid(GABA) in the rat hippocampus. Real-time fluorescence quantitative PCR(qRT-PCR) and Western blot were employed to assess the gene and protein expression of ionotropic glutamate receptors in the hippocampal region. Transmission electron microscopy was utilized to observe the changes in the ultrastructure of synaptic neurons in the hippocampal region. Long-term potentiation(LTP) detection technique was employed to record the changes in population spike(PS) amplitude in the hippocampal region of mice in each group. The behavioral results showed that compared with the model group, the Suanzaoren Decoction group effectively increased the number of entries into open arms, time spent in open arms, percentage of time spent in open arms out of total movement time, number of entries into open arms out of total entries into both arms(P<0.01), and significantly increased the time spent in the light box and the number of shuttle crossings(P<0.01). There was an increasing trend in the number of grid crossings, entries into the center grid, and time spent in the center grid, indicating a significant anxiolytic effect. ELISA results showed that compared with the model group, the Suanzaoren Decoction group exhibited significantly reduced levels of Glu, Glu/GABA ratio(P<0.01), and significantly increased levels of GABA(P<0.01) in the rat hippocampus. Furthermore, Suanzaoren Decoction significantly decreased the gene and protein expression of NMDAR(GluN2B and GluN2A) and AMPAR(GluA1 and GluA2) compared with the model group. Transmission electron microscopy results demonstrated improvements in synapses, neuronal cells, and organelles in the hippocampal region of the Suanzaoren Decoction group compared with the model group. LTP detection results showed a significant increase in the PS amplitude changes in the hippocampal region of Suanzaoren Decoction group from 5 to 35 min compared with the model group(P<0.05, P<0.01). In conclusion, Suanzaoren Decoction exhibits significant anxiolytic effects, which may be attributed to the reduction in NMDAR and AMPAR expression levels and the improvement of synaptic plasticity.
Rats ; Mice ; Animals ; Receptors, Ionotropic Glutamate/metabolism* ; Hippocampus ; Neuronal Plasticity ; Receptors, N-Methyl-D-Aspartate/genetics* ; Anxiety/genetics* ; gamma-Aminobutyric Acid

Natural products are an important source for the development of antitumor lead compounds, but the pharmacological effects and regulatory mechanisms of natural products in osimertinib resistance in non-small cell lung cancer (NSCLC) are not well understood. The natural product ligustroflavone was used as the research object to analyze its efficacy in osimertinib-resistant NSCLC cells by cell proliferation assay and cell cycle detection. The potential targets of ligustroflavone in osimertinib-resistant NSCLC cells were screened by public databases and bioinformatics, molecular docking and microscale thermophoresis were used to identify the interaction between privet and target molecules. Western blot was used to detect the effect of privet on the target molecules and their downstream pathways. Ligustroflavone reduced the proliferation of osimertinib-resistant NSCLC cells, and could arrest the cell cycle. Cyclin-dependent kinase 6 (CDK6) was the potential target of ligustroflavone in osimertinib-resistant NSCLC cells. Ligustroflavone inhibited the activation of CDK6-Rb axis. Together, ligustroflavone could regulate osimertinib resistance in NSCLC cells by binding cell cyclin-related molecules. This study provides a theoretical basis for the targeted drug resistance of NSCLC with natural products, and also provides a new idea for the development of clinical drug combination.

Nucleocytoplasmic transport is the basic cellular activity of eukaryotic cells, which plays a role in cell physiological and pathological processes. A large amount of evidences indicate that impaired nucleocytoplasmic trafficking has emerged as a mechanism contributing to the pathology of neurodegenerative diseases. The regulation of nucleocytoplasmic transport is crucial to elucidate the pathogenesis and intervention in the neurodegenerative diseases. This article summarizes the evidences in disturbed nucleocytoplasmic transport of neurodegenerative diseases in the past two decades, further explores the directions and provides a theoretical basis for the pathogenesis and drug targets in neurodegenerative diseases.

OBJECTIVE: To establish the occupational exposure limit for trimethyltin chloride(TMT) in workplace air. METHODS:According to the GBZ/T 210.1-2008 Guide for Establishing Occupational Health Standards--Part 1: Occupational Exposure Limits for Airborne Chemicals in the Workplace, the relevant literatures on toxicology, population epidemiology and foreign occupational exposure limit of TMT were collected and analyzed. A total of 276 workers with TMT occupational exposure were selected as the exposure group and 25 workers without TMT occupational exposure were selected as the control group.Worksite survey of occupational health and occupational medical examination were carried out. Combined with the literature data, the occupational exposure limit of TMT in the workplace air was calculated by using the 90% medical reference level(internal exposure limit) of the urine TMT level of workers who exposed to TMT without moderate hypokalemia. RESULTS: The time-weighted average of TMT in the workplace air is 0.100 mg/m~3 and the short-term exposure limit is 0.200 mg/m~3 in the United States based on total organic tin. The highest concentration of TMT in the workplace air in Germany is 0.005 mg/m~3. The literature data analysis results showed that the incubation period of TMT poisoning is mostly 3-6 days, and the main symptoms of TMT poisoning are hypokalemia in the early stage, followed by neuropsychiatric symptoms such as headache, memory loss and aggressive behavior. The median(M) and the 0-100 th percentile(P_0-P_(100)) of exposure to TMT were 8.35(< 0.20-91.40) μg/m~3 in the exposure group. The individual TMT exposure level of workers in different positions from high to low were crushing, granulation, withdrawal and assembly positions. The M(P_0-P_(100)) of urinary TMT level in the exposure group was 16.94(<0.50-591.14) μg/L. There was a positive correlation between the individual TMT exposure level and urine TMT level in the exposure group(Spearman correlation coefficient=0.62, P<0.01). The detection rate of hypokalemia in the exposure group was higher than that in the control group(26.1% vs 4.0%, P < 0.05). However, there was no significant difference in the detection rate of moderate hypokalemia between the two groups(3.3% vs 0.0%, P>0.05). The 90% medical reference value of urine TMT was 89.90 μg/L in workers exposed to TMT without moderate hypokalemia. CONCLUSION: In order to prevent acute hypokalemia damage caused by TMT, we recommended that the occupational exposure limit of TMT in the workplace air should be set at 0.025 mg/m~3 in China, and this limit should be the maximum allowable concentration.

Objective: To investigate the mRNA and protein expressions of outer dense fiber 2 (ODF2) in the sperm of the asthenospermia patient and their differences from those in normal healthy men. METHODS: According to the WHO criteria, we collected semen samples from 45 asthenozoospermia patients and 15 normal healthy volunteers. Using computer-assisted sperm analysis (CASA), we divided the semen samples from the asthenospermia patients into a mild, a moderate and a severe group, and determined the mRNA and protein expressions of ODF2 in different groups by RT-PCR and Western blot. RESULTS: Compared with the normal healthy men, the expression of the ODF2 gene showed no statistically significant difference in the mild asthenospermia group (1.112 0 ± 0.525 5 vs 0.688 0 ± 0.372 0, P >0.05) but remarkably decreased in the moderate (0.483 3 ± 0.186 3, P <0.05) and severe asthenospermia patients (0.448 3 ± 0.340 8, P <0.01). The OD value (ODF2/β-actin) of the ODF2 protein in the normal men exhibited no statistically significant difference from that in the mild asthenospermia group (0.458 7 ± 0.052 1 vs 0.326 1 ± 0.071 4, P >0.05), but markedly lower than in the moderate (0.145 4 ± 0.053 6, P <0.05) and severe asthenospermia patients (0.122 7 ± 0.045 7, P <0.01), which was consistent with the results of RT-PCR. CONCLUSIONS Decreased mRNA and protein expressions of ODF2 in the sperm are positively correlated with declined sperm motility of the asthenospermia patient, which is suggestive of the involvement of the ODF2 gene in the regulation of sperm motility.
Asthenozoospermia ; metabolism ; physiopathology ; Case-Control Studies ; Down-Regulation ; Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Male ; RNA, Messenger ; metabolism ; Semen Analysis ; Sperm Motility ; Sperm Tail ; Spermatozoa ; metabolism

The aim of this study was to investigate if transfusion of mesenchymal stem cells (MSC) could exhibit beneficial effects on rheumatoid arthritis. Human bone marrow MSC were intraperitoneally injected into Wistar rats with collagen-induced arthritis at a dose of 10(7) on the next day (preventive group) or 2 weeks (treatment group) after collagen II induction, once a week for 2 weeks (preventive group) or 4 weeks (treatment group). The control group was given normal saline (NS) at corresponding time. The symptom scorings were documented weekly from the second week of the induction. On week 6, the hind joints of the rats were pathologically examined and the activation status of splenocytes was analyzed by flow cytometry. The results showed that all the rats developed arthritis and subsequent joint abnormality. On the sixth week, symptom scores of the rats that received MSC preventive (9.5 ± 0.5) or therapeutic (9.4 ± 0.6) infusions had no significant difference between each other, but were significantly greater than those of the NS controls (7.6 ± 0.6, P < 0.05). Consistently, pathological examination on the involved knees showed that the synovitis and arthritis scorings of MSC treated rats were greatly elevated compared with NS controls. Furthermore, the ratios of CD86(+) cells in the spleens of MSC prevention, MSC treatment and NS control groups were (4.16 ± 1.48), (4.06 ± 1.97) and (4.15 ± 2.04) respectively, while those of CD11b/c(+)CD86(+) cells were (1.04 ± 0.68), (0.95 ± 0.56) and (0.98 ± 0.44), all of which were significantly higher than those of healthy controls [(0.97 ± 0.18) and (0.30 ± 0.17), P < 0.05 for both parameters]. It is concluded that MSC infusion has little beneficial effects on collagen-induced arthritis in rats, conversely, MSC therapy aggravated the damage of the involved joints, its underlying mechanisms need to be further investigated.
Animals ; Arthritis, Experimental ; pathology ; prevention & control ; therapy ; Bone Marrow Cells ; Cells, Cultured ; Humans ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Wistar ; Transplantation, Heterologous ; Treatment Outcome

OBJECTIVETo evaluate the feasibility and effectiveness of needle and syringe exchange program among a community of injecting drug users (IDUs) on AIDS prevention.

METHODSA quasi-experiment design was used in a controlled community intervention study. Needle and syringe exchange program was implemented for 10 months in IDUs of an intervention community, including peer education and health education, provision of free needles and syringes, and collecting back of used needles and syringes by trained peer educators and local health workers, whereas no intervention measure in a control community was instituted. Interviews with IDUs were conducted before and after intervention with a snowballing strategy to evaluate its effectiveness.

RESULTSA total of 428 and 429 IDUs were interviewed with structured questionnaire before and after intervention in intervention and control communities, respectively. Results revealed that awareness of HIV-related knowledge increased from 29.4% to 58.7% in the intervention community. Multivariate logistic regression analysis showed that awareness of HIV-related knowledge was higher in those who had read health education materials (OR = 2.93, 95% CI 2.12 - 4.04). As compared with the baseline data, frequency of sharing needles and syringes in past 30 days in the intervention community decreased from 48.9% to 20.4% in before intervention community (chi(2) = 41.02, P = 0.001), whereas there was no significant change in the control community. The causes of sharing needles and syringes in the intervention community included 'disable to get needle and syringe during the night', 'lack of needle and syringe when injecting at friend's home', 'not daring to buy needle and syringe for fear of being arrested' and 'no money to buy needle and syringe', declined markedly.

CONCLUSIONSNeedle and syringe exchange program was feasible and effective in reducing their risky drug injecting behavior among IDUs in communities. Such strategy should be adopted in the country to reduce rapid spread of HIV.

Adult ; China ; epidemiology ; Female ; HIV Infections ; prevention & control ; transmission ; Health Education ; Humans ; Male ; Needle Sharing ; adverse effects ; statistics & numerical data ; Needle-Exchange Programs ; economics ; organization & administration ; Pilot Projects ; Program Evaluation ; Substance Abuse, Intravenous ; complications ; epidemiology