Objective: To examine whether deuterium depleted environment may affect the biological features of human gastric cancer cells(SGC-7901)and explore the possible underlying mechanisms.Methods: SGC-7901 cells were cultured in RPMI-1640 medium prepared with distilled water of different deuterium concentrations(experimental group:25ppm deuterium;control group:150ppm deuterium). Assays on cellular proliferation, cell cycle and apoptosis were conducted at different time points and comparison. The protein expression of proliferating cell nuclear antigen (PCNA) was measured using Western blot.Results: In contrast to 150ppm group, the proliferation rate of SGC-7901 cells in 25ppm deuterium was decreased by 10% as indicated by the CCK-8 assay. Wound healing ability and the colony formation ability of these cells were also significantly suppressed (P<0.05). Flow cytometry analysis further revealed that exposure to 25ppm significantly increased the ratio of cancer cells at G1 phase (P<0.01) while decreased the ratio at S phase (P<0.05) compared to the 150ppm group. There was no significant difference in apoptosis between the two groups. Down-regulated expression of PCNA was also identified in cancer cells treated with 25ppm deuterium.Conclusion: Deuterium depleted environment inhibited the proliferation of gastric cancer cells, which may be attributed to the down-regulation of PCNA and cell cycle arrest at G1 phase.

Objective: To explore the effects of SR59230A on the tension and microRNA (miRNA) expression of rat thoracic aorta.Methods: Forty-four SD rats were used in the experiment. Twenty-four rats were used to observe the effect of SR on the tension of thoracic aortic rings. Another 20 rats were randomly divided into control (n=10) and SR group(n=10). Rats in SR group were injected SR intraperitoneally,and in control group were given 0.9% of saline. After 5 weeks, the blood pressure of all rats were measured. Then the tension to NA and the expression of miRNA of thoracic aorta rings were measured.Results: (1) The tension of thoracic aortic rings responding to 30 mmol/LKCl were increased by pretreatment of SR (P<0.05); (2) After 5 weeks injection of SR, systolic pressure was increased (P<0.05); (3) The tension in SR group was increased in presence of 1 μmol/L and 10 μmol/L of NA (P<0.05,P<0.01). (4) After 5 weeks of SR in vivo application,18 miRNA were down-regulated, 7 of them had statistical significance, they were rno-miR-143-3p, rno-miR-29b-3p, rno-miR-31a-5p, rno-let-7b-5p, rno-miR-214-3p, rno-miR-222-3p and rno-miR-352; 11 miRNA were up-regulated, 4 of them had statistical significance, they were rno-miR-206-3p、rno-miR-223-3p、rno-miR-342-3p and rno-miR-499-5p respectively.Conclusion: SR59230A increased the tension of rat thoracic aorta. In vivo administration of SR led to increase of systolic pressure of rat,down-regulation of rno-miR-143-3p、rno-miR-29b-3p、rno-miR-31a-5p、rno-let-7b-5p、rno-miR-214-3p、rno-miR-222-3p、rno-miR-352 and up-regulation of rno-miR-206-3p、rno-miR-223-3p、rno-miR-342-3p and rno-miR-499-5p.

Objective: To investigate the protective effect of curcumin analogue L6H4 on the kidney from the type 2 diabetic rats.Methods: Twenty-four SPF male SD rats were randomly divided into 3 groups(n=8):normal control group(NC),diabetes mellitus group(DM) and DM+L6H4-treatment group(DT). After rats were fed with high-fat diet for 4 weeks, both the DM and DT groups were injected with streptozotocin intraperitoneally to induce type 2 diabetes mellitus models. The rats in DT group were given L6H4 by gavage at the dose of 0.2 mg/kg·d for 8 weeks. After the treatment, the 24 h urinary protein, fasting blood glucose (FBG), triglyceride (TG), serum creatinine(Scr),blood urea nitrogen (BUN) and uric acid (UA) were detected biochemically. The pathological changes of the kidneys were observed under light and transmission electron microscopes. The expressions of TGF-β1, FN and Col IV were detected by immunohistochemistry.Results: The levels of the 24 h urinary protein, FBG, TG, Scr and BUN were elevated significantly in diabetic group(P<0.01). The glomerular volume of DM group rats became irregularly enlarged, diffused mesangial matrix accumulated, with basal membrane proliferous hypertrophy and fusion phenomenon of foot process, the expressions of TGF-β1,FN and Col-IV were elevated significantly (P<0.05). After treated with L6H4, the levels of the 24 h urinary protein, FBG, TG, Scr and BUN were decreased in DT group compared to DM group (P<0.01), the morphological changes of kidney were ameliorated. The expression levels of TGF-β1, FN and Col-IV were downregulated (P<0.05).Conclusion: L6H4 exerts the protective effect on kidneys of type 2 diabetic rats by reducing expression of TGF-β1, inhibiting secretion of Col-IV and FN, relieving the deposition of the extracellular matrix.

Objective: To observe the effects of calcitonin gene-related peptide (CGRP) on eukaryotic translation initiation factor 3a (eIF3a) and p27 expression in bleomycin-induced pulmonary fibrosis of rats and its possible mechanism.Methods: Twenty-four male SD rats weighing 180~220 g were randomly divided into three groups (n=8):control group, bleomycin group, bleomycin plus capsaicin group. Bleomycin (5 mg/kg) was used to induce pulmonary fibrosis rat model. Rats were given capsaicin (50 mg/kg·d) by subcutaneous injections 4 days before to deplete endogenous CGRP. At the end of experiments, blood samples were collected from carotid artery to determinate the plasma levels of CGRP by ELISA. The cells were divided into 6 groups as follows:control group, transforming growth factor-β1 (TGF-β1) group, +CGRP (1, 10, 100 nmol/L) group, +CGRP 100 nmol/L and CGRP8-37 1 μmol/L group respectively(n=9). TGF-β1 (5 ng/ml) stimulated proliferation of pulmonary fibroblasts and proliferation was measured by BrdU marking. The expression levels of eIF3a, p27, α-smooth muscle actin (α-SMA) and collagen Ⅰ were detected by immunohistochemisty, real-time PCR or Western blot.Results: The expressions of eIF3a, α-SMA, and collagen I were increased and the expression of p27 was decreasing in pulmonary fibrosis rats induced by bleomycin. Exogenous application of CGRP significantly inhibited TGF-β1-induced proliferation and differentiation of pulmonary fibroblasts and the expressions of α-SMA, collagen I and eIF3a, and upregulated the expression of p27. All these effects of CGRP were abolished in the presence of CGRP8-37.Conclusion: These results suggest that endogenous CGRP is related to the development of pulmonary fibrosis induced by bleomycin, and the inhibitory effect of CGRP on proliferation of lung fibroblasts involves the eIF3a/p27 signaling pathway.

Objective: To observe the effects of arecoline on lipid metabolism in 3T3-L1 adipocytes and explore its possible mechanisms.Methods: 3T3-L1 pre-adipocytes were induced into adipocytes with the classic "cocktail" method, subsequently, adipocytes were treated with arecoline at the concentrations of 0, 25, 50 and 100 μmol/L for 72 hours. After 72 hours, cell vability was measured with MTT method, lipid droplet accumulation in the cytoplasm was observed with oil red O staining, the protein expression of fatty acid synthase (FAS), adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) were detected by Western blot assay.Results: There were a large number of lipid droplets in the cytoplasm in the differentiated 3T3-L1 adipocytes. MTT results showed that 0~100 μmol/L arecoline had no significant effect on cell vability; oil red O staining found arecoline reduced lipid amount in 3T3-L1 adipocytes; Western blot results showed that compared with 0 μmol/L arecoline group (the control group), arecoline significantly reduced the protein level of FAS and increased the protein levels of ATGL and HSL, and 50 μmol/L arecoline group was the most significant.Conclusion: Arecoline significantly increased lipolysis of 3T3-L1 adipocyte, which might be associated with decreased the FAS expression of key enzyme of lipid synthesis and increased the ATGL and HSL expression of key enzyme of adipolysis.

Objective: To observe the effects of 5-HT_1Areceptor antagonist p-MPI on ethanol induced hypothermia and behavioral ther-moregulatory response in rats.Methods: Core temperature and motor activities were monitored in undisturbed male SD rats using radioteleme-try. The behavioral thermoregulatory response and core temperature were monitored in rats using radiotelemetric temperature gradient apparatus. The rats were placed in a temperature gradient that permitted the selection of ambient temperature ranging from 15℃ to 40℃. Effect of ethanol (3 g/kg) and 5-HT_1A receptor antagonist p-MPPI(1 mg/kg) on core temperature, motor activities, and the behavioral thermoregulatory re-sponse were observed in rats.Results: ①Ethanol led to a rapid reduction in core temperature. The hypothermic responses were accompanied with a preference for cooler ambient temperature. ②5-HT_1A receptor antagonist attenuated the hypothermia induced by ethanol, and accompa-nied with a selection for warmer ambient temperature.Conclusion: ①Behavioral thermoregulatory observations suggested that the ethanol could decrease the thermoregulatory set point,because rats treated with ethanol selected cooler ambient temperature facilitates the reduction in core temperature.②5-HT might be involved in ethanol-induced hypothermia and behavioral thermoregulatory response.

Objective: To observe the effects of aerobic exercise on activity of extracellular signal-regulated kinase (ERK1/2) in skeletal muscle of type 2 diabetic rats and explore the prevention and control mechanism of aerobic exercise on type 2 diabetes.Methods: Seventy five SD rats were randomly divided into:normal control group(CON) including 15 rats was fed with normal diet, diabetes control group 1(DC1), diabetes exercise group 1(DE1), diabetes control group 2(DC2), diabetes exercise group 2(DE2). Diabetes model group were fed with high-fatty and high-sugar diet. The diabetes model rats were fed with high-fatty and high-sugar diet for 8 weeks,.Diabetes group 2 rats were injected intraperitoneal streptozotocin(STZ)to induce type 2 diabetes. At the early stage of last swimming week, diabetes exercise group1 and diabetes control group 1 were injected with STZ (35 mg/kg) at the same time, After three days, if the level of blood glucose was ≥ 16.7mmol/L, the model was successful. After 8 week-interventions, all the rats were killed, the serum levels of insulin and the expression of ERK1/2 protein in skeletal muscle were determined.Results: ①Compared with the normal control group, the levels of total cholesterol(TC), triglyceride(TG), low density lipoprotein cholesterin(LDL-C), free fatty acid(FFA), fasting blood-glucose(FBG), fasting insulin(FIN) and insulin resistance index(HOMA-IR) were increased significantly in diabetes control group(P<0.05 or 0.01). However, the expression of ERK1/2 phosphorylation protein was decreased obviously in diabetes control group. The content of ERK1/2 protein was decreased obviously in diabetes control group 2 had (P<0.05). 2.After eight weeks' swimming, compared with the diabetes control group, the levels of TC,TG, FFA, LDL-C, FBG, FIN and HOMA-IR were decreased significantly in diabetes exercise group(P<0.05 or 0.01). At the same time, the expression of ERK1/2 phosphorylation protein was increased obviously in diabetes exercise group(P<0.05).Conclusion: Long-term aerobic exercise can improve the skeletal muscle ERK1/2 phosphorylation and insulin resistance of type 2 diabetic rats, thereby lowering blood glucose. It is probably one of the mechanisms to improve glucose metabolism disorders and insulin sensitivity.

Objective: To investigate the roles of change in sodium pump activity and endoplasmic reticulum stress (ERS) in ischemia/reperfusion (I/R) injury of the isolated rat hearts.Methods: Sixty male SD rats were randomly divided into 6 groups (n=10 each):normal control group (NC), I/R group (I/R), ouabain-I/R group (OUA-I/R), anti-digoxin antiserum-I/R group (Anti-Dig-I/R), PP2 (Src kinase inhibitor)-ouabain-I/R group (PP2-OUA-I/R),U73122 (PLC inhibitor)-ouabain-I/R group (PP2-OUA-I/R). The isolated rat hearts were perfused on the Langendorff apparatus. Except for NC group, all the hearts were subjected to 30 min global ischemia and followed by 120 min reperfusion. The cardiac function indexes were recorded at the same time. The coronary effluent was collected for estimating the activity of lactate dehydrogenase (LDH) and creatine kinase (CK). The activity of Na⁺-K⁺-ATPase and intracellular calcium concentration in myocardial tissue were measured. Apoptosis was evaluated by Flow cytometric analysis. The expressions of sodium pump α1 subunit, glucose regulated protein(GRP78),C/EBP homologous protein (CHOP) and Bcl-2/Bax were determined by Western blot.Results: Pretreatment with ouabain significantly reduced the recovery of cardiac function, increased the levels of CK, LDH and intracellular calcium concentration, decreased the activity of Na⁺-K⁺-ATPase. In addition, ouabain markedly increased the myocardial apoptosis index, down-regulated the expressions of sodium pump α1 subunit and Bcl-2, up-regulated the expressions of GRP78,CHOP and Bax; while these changes were significantly improved in the Anti-Dig-I/R group compared with those in the I/R group; PP2 or U73122 partially blocked the effects of ouabain on myocardial I/R injury. Compared with the OUA-IR group, the recovery of cardiac function, the activity of Na⁺-K⁺-ATPase and the expressions of sodium pump α₁ subunit and Bcl-2 were significant higher, meanwhile the leakage of CK and LDH, intracellular calcium concentration, myocardial apoptosis index and the expressions of GRP78 and Bax were significantly lower in PP2-OUA-I/R and U73122-OUA-I/R group.Conclusion: Changes in sodium pump function and endoplasmic reticulum stress all participate in the process of I/R injury. Current findings further suggest that sodium pump mediates ERS by activating signals of Src and PLC pathway, which may be one of the mechanisms of apoptosis induced by I/R injury.

Objective: To investigate the expression of mRNA and protein of Calcium activated chloride channel (CLCA2) in hypoxic pulmonary artery smooth muscle cell (PASMCs) of rat and it's relationship with ERK1/2 signal pathway.Methods: PASMCs were randomly divided into 5 groups including normal group(N group), hypoxia group(H group), DMSO group(D group), U0126 group (U group) and Staurosporine aglycone group(SA group). The protein expression of CLCA2 in PASMCs was detected by Western blot.The mRNA expression of CLCA2 was detected by half quantitative reverse transcription polymerase chain reaction (RT-PCR).Results: The mRNA and protein expressions of CLCA2 in H group were significantly higher than N group (P<0.01). Comparing with D group,the mRNA and protein expressions of CLCA2 were significantly increased in U group (P<0.01),the mRNA expression of CLCA2 in SA group was obviously decreased (P<0.01) with slightly decreasing of its protein expression.Conclusion: Hypoxia promotes the expressions of mRNA and protein of CLCA2 in rat PASMCs. The ERK1/2 pathway activator Staurosporine aglycone reduces the mRNA and protein expression of CLCA2 in rats PASMCs and the ERK1/2 pathway inhibitor U0126 induces the upregulation of the mRNA and protein expressiosn of CLCA2 in rats PASMCs.

Objective: To investigate the effects of Shiquanyuzhentang (Traditional Chinese Medicine) on the immunologic function of tu-mor-bearing mice and its mechanism of antitumor.Methods: Thirty SPF grade male Kunming mice transplanted with H₂₂ hepatocellular carci-noma were divided into three groups randomly:model group,positive control group and Shiquanyuzhentang group(n=10). Another 10 mice were selected as normal control group. Normal control group and model group received normal saline and distilled water supplementation by 10 mL/kg everyday. Positive control group and Shiquanyuzhentang group received Shengyi(80 mg/ml)and Shiquanyuzhentang decoction at the doses of 8 g/kg and 18 g/kg respectively everyday. After 14 days of continuous administration, the mice were killed and the thymus, spleen index, tumor inhibition rate,peripheral blood leukocytes,lymphocyte content,cell percentage of T cell subsets CD3, CD4, CD8,interleukin 2 (IL-2)in serum,tumor necrosis factor-α(TNF-α),interferon β(IFN-β) content,lymphocyte proliferation ability and NK cell were measured.Results: Compared with normal control group, the weight of mice and thymus and spleen index of the Shiquanyuzhentang group were increased significantly(P<0.05);leukocyte and lymphocyte CD3、CD4、CD8 and TNF-α were increased greatly(P<0. 05),IL-2 and IFN-β were de-creased significantly(P<0.05). Compared with model group,thymus and spleen index of Shiquanyuzhentang group were increased significant-ly(P<0.05);leukocyte and lymphocyte CD3、CD4、IL-2、IFN-β and TNF-α of Shiquanyuzhentang group were increased significantly(P<0. 05),CD8 content was decreased significantly(P<0.05);NK cell and lymphocyte proliferation were increased significantly(P<0.05).Conclusion: Shiquanyuzhentang can promote the growth of immune organs in mice bearing H₂₂, enhance immune function and is beneficial to the recovery of tumor body.

Objective: To observe the change of angiotensin converting enzyme (ACE) and ACE2 in the murine myocardium followed hemorrhagic shock and the role of post-hemorrhagic shock mesenteric lymph (PHSML) drainage.Methods: Twenty-four male mice were ran-domly divided into control, sham, shock, and shock + drainage groups. A hemorrhagic shock model was established and then fluid resuscita-tion was performed to the mice in the shock and shock + drainage groups, and the PHMSL was drained in the shock + drainage group after fluid resuscitation. After 6 h of resuscitation in the shock and shock + drainage groups or corresponding time in the sham group, or after anesthesia in the control group, the myocardial tissues were harvested for the determination of the mRNA expressions of ACE, ACE2, angiotensin Ⅱ (Ang Ⅱ) type 1 receptor (AT1R), and Mas related G protein coupled receptor (Mas1R) using the method of qRT-PCR, and the levels of Ang Ⅱ and Ang (1-7) using the method of ELISA.Results: In the myocardial tissue of shock group, the ACE and AT1R mRNA expressions and Ang Ⅱ level were significantly increased than those of the control and sham groups, the ACE2 and Mas1R mRNA expressions were significantly de-creased than that of the control and sham groups, the Ang (1-7) level was decreased compared with the control group, the ratios of ACE/ACE2, Ang Ⅱ/Ang (1-7), and AT1R/Mas1R in the shock group were significantly increased than the control and sham groups. Meanwhile, PHSML drainage obviously suppressed the effects of hemorrhagic shock on these indices.Conclusion: Hemorrhagic shock up-regulated the ACE-Ang Ⅱ-AT1R axis, down-regulated the ACE2-Ang (1-7)-Mas1R axis, and induced the unbalance of ACE and ACE2 in myocardial tis-sue. PHSML drainage decreased the ACE-Ang Ⅱ-AT1R axis and increased the ACE2-Ang (1-7)-Mas1R axis, resulted in the balance of ACE and ACE2.

Objective: To investigate the interactive effects of different temperatures and ambient PM2.5 on the rat alveolar macrophages.Methods: The rat alveolar macrophages were collected. The cells were exposed in vitro to 18℃, 24℃, 30℃, 37℃ and 43℃ with PM2.5 at the concentrations of 100 μg/ml, 50 μg/ml, 25 μg/ml and 0 μg/ml respectively. The cells were cultured in the different cases for 8 hours, then cytotoxicity was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)reduction assay and phagocytosis function of macrophages was assessed by neutral red absorption test.Results: The relative survival rate and the cytophagocytic function of alveolar macrophages of rats among the different concentration groups decreased significantly (P<0.05) compared with the blank control group. Both were dose-dependent. The 37℃ group had the highest level of relative survival rate and the cytophagocytic function compared with other different temperatures groups. Interactive effect of different temperatures and ambient PM2.5 was not observed. But the lower temperature and the higher PM2.5 concentration group had stronger toxicity to alveolar macrophages.Conclusion: The results suggested that different temperatures and ambient PM2.5 have cytotoxicity on alveolar macrophages,injuring the phagocytosis. The two factors had some interaction.

Objective: To investigate the effects of long non-coding RNA SPRY4-IT1 (LncRNA) on proliferation and metastasis of medul-loblastoma cells.Methods: SPRY4-IT1siRNA and control fluorescence siRNA were transfected into medulloblastoma cell line Daoy with Lipo-fe ctamine 2000 and were divided into control group and si-SPRY4-IT1 group. Relative expression of SPRY4-IT1 mRNA were detected by real-time quantitative PCR. The change of cell proliferation were examined using CCK-8 kit and clone forming experiment. The change of cell inva-sion and metastasis were examined by matrigel invasion assay and cell metastasis experiment respectively, The expression of matrix metallopro-teinase MMP-2 and MMP-9 were detected by Western blot.Results: In si-SPRY4-IT1 group,the SPRY4-IT1 mRNA expression level, cell pro-liferation in vitro,cell invasion and migration ability, MMP-2 protein expression were significantly lower than those in the control group.Conclusion: Interference SPRY4-IT1 expression has prominent inhibitory effect on the cell proliferation、invasion and metastasis of medulloblastoma cell line Daoy.

Objective: To detect the expression changes of proton-sensing receptor G2 accumulation (G2A) and ovarian cancer G protein-coupled receptor 1(OGR1) in human peripheral blood cells in hypoxic pulmonary hypertension patients(HPH).Methods: Thirty-one patients with HPH were enrolled for HPH group(16 men and 15 women,age:(65.19±5.86) and thirty healthy persons were enrolled for the control group (NC group). The peripheral blood samples were collected and the mRNA expressions of G2A and OGR1 were determined by using real-time fluorescent quantitative PCR. The serum levels of tumor necrosis factor-α (TNF-α) were detected by using enzyme-linked immunosorbent assay (ELISA).Results: PaCO₂ was increased significantly in HPH group than that of the NC group (P<0.05). Forced expiratory volume in 1 sencond(FEV1)pro% and FEV1/forced vital capacity(FVC)in HPH group were significant lower than those of the NC group(P<0.05). The expressions of peripheral blood G2A mRNA and TNF-α in HPH group were increased dramatically than those of the NC group(P<0.05). The expressions of OGR1 mRNA in peripheral blood had no difference between HPH group and NC group. The expressions of G2A and TNF-α in HPH group were positively related to pulmonary artery pressure significantly.Conclusion: The expression of proton-sensing receptor G2A and the level of TNF-α are increased in peripheral blood cells of patients with pulmonary hypertension.The expressions of TNF-α,G2A and pulmonary artery pressure have positive correlation in the HPH group.

Objective: To offer a series of efficient methods to physiological researchers in following the four principles of experimental de-sign (hereinafter referred to as four principles).Methods: We expounded the concrete way of following the four principles through giving an outline of the four principles, elaborating the common mistakes and cases in the applications of four principles in physiological research and in-troducing briefly how to follow the four principles in physiological research.Results: We should choose the appropriate randomization method according to the actual situation, usually stratified randomization was a good way. We should strive to set reasonable control group to reflect the value of control principle veritably. Giving the appropriate sample size for each group rightly was the basic guarantee for the research result could be reproduced. Following the principles of randomized, controlled and repetition strictly could achieve an excellent balance between con-trol groups ultimately, that was to say that the balanced principle was a principle of gatekeeper.Conclusion: Following the four principles strictly is critically important to assure the result accurately and reliably in physiological research.