Experimental study of Lentinan antagonizes against hepatic lipid deposition induced by sodium arsenite in mice / 公共卫生与预防医学

Objective To investigate the ameliorative effect of Lentinan (LNT) on sodium arsenite (SA)-induced hepatic lipid deposition in mice. Methods C57BL/6 mice were used as the experimental subjects, which were divided into control group, SA-exposed group, LNT + SA-exposed group and LNT control group. Blood and liver tissue samples were collected at the end of the experiment, and serum glutathione transaminase (ALT) and glutathione aminotransferase (AST) levels were detected by enzyme-linked immunosorbent assay (ELISA). A part of liver tissues was stained with hematoxylin-eosin (HE) or oil red O to observe the characteristics of liver pathological damage and lipid deposition, and another part of liver tissues was used to detect triglyceride (TG) and Adiponectin (APN) levels by ELISA. Results Compared with control group or LNT control group, SA-exposed group showed the increased levels of AST and ALT, showing the characteristics of liver histopathological damage and lipid deposition, and the APN level decreased while the TG level increased (P<0.05). Compared with SA-exposed group, the levels of AST and ALT decreased in LNT + SA-exposed group, showing the reduced degree of liver tissue damage and lipid deposition, and APN level upregulated while TG level downregulated (P<0.05). Conclusion Chronic SA exposure induces liver function damage, APN downregulation and lipid deposition in C57BL/6 mice, while LNT intervention leads to the significantly improvement of hepatic damage and lipid deposition, which may be related to the elevated APN level in liver.

Effect of Ginkgo biloba extract on improving hepatic insulin resistance induced by arsenic exposure based on network pharmacology / 环境与职业医学

Background Arsenic exposure is a common and important environmental and occupational hazardous factor in China, and arsenic-induced insulin resistance (IR) has attracted widespread attention as a negative health outcome to the population. Objective To explore part of the mechanism of hepatic IR induced by arsenic exposure based on the peroxisome proliferators-activated receptors γ (PPARγ)/ glucose transporter 4 (GLUT4) pathway, and to investigate potential effects of Ginkgo biloba extract (GBE) on hepatic IR induced by arsenic exposure and associated mechanism of action. Methods The target of drug action was predicted by network pharmacology and verified by in vivo and in vitro experiments. In vivo experiments: 48 SPF C57BL/6J male mice were divided into 4 groups, including control group, 50 mg·L−1 NaAsO2 model group (NaAsO2), 50 mg·L−1 NaAsO2+10 mg·kg−1 GBE intervene group (NaAsO2+GBE), and 10 mg·kg−1 GBE group (GBE), 12 mice in each group. The animals were given free access to purified water containing 50 mg·L−1 NaAsO2, or given intraperitoneal injection of normal saline containing 10 mg·kg−1 GBE once per week. After 6 months of exposure, blood glucose detection, intraperitoneal glucose tolerance test (IPGTT), and insulin tolerance test (ITT) were performed. Serum and liver tissues were collected after the mice were neutralized, liver histopathological sections were obtained, serum insulin levels, liver tissue glycogen content, glucose content were detected by enzyme linked immunosorbent assay (ELISA), and the expression of PPARγ and GLUT4 proteins was detected by Western blot (WB). In vitro experiments: HepG2 cells were divided into 4 groups, including control group, 8 μmol·L−1 NaAsO2 group (NaAsO2), 8 μmol·L−1 NaAsO2 + 200 mg·L−1 GBE intervene group (NaAsO2+GBE), and 200 mg·L−1 GBE group (GBE). The levels of glycogen and glucose were detected by ELISA, and the expression of PPARγ and GLUT4 proteins was detected by WB. Results A strong binding effect between GBE and PPARγ was revealed by network pharmacology. In in vivo experiments, the NaAsO2 group exhibited an elevated blood glucose compared to the control group, and the NaAsO2+GBE group showed a decreased blood glucose compared to the NaAsO2 group (P<0.01). The histopathological sections indicated severe liver structural damage in the arsenic exposure groups (NaAsO2 group and NaAsO2+GBE group), with varying staining intensity, partial liver cell necrosis, and diffuse red blood cell appearance. Both results of in vitro and in vivo experiments showed a decrease in glycogen synthesis and glucose uptake in the NaAsO2 groups compared to the control groups, which was alleviated in the NaAsO2+GBE group (P<0.01). The results of WB revealed inhibited PPARγ expression and reduced GLUT4 levels on the cell membrane, and all these changes were alleviated in the NaAsO2+GBE group (P<0.01). Conclusion This study findings suggest that GBE antagonizes arsenic exposure-induced hepatic IR by regulating the PPARγ/GLUT4 pathway, indicating that GBE has a protective effect on arsenic exposure-induced hepatic IR, and PPARγ may be a potential therapeutic target for arsenic exposure-induced hepatic IR.

The Effect of Ferroptosis on the Activation of Human Hepatic Stellate Cells Induced by Inorganic Arsenic / 医学研究杂志

Objective To investigate the effect of NaAsO2 on ferroptosis in human hepatic stellate cells(LX-2).Methods LX-2 cells were cultured in vitro,and different concentrations of NaAsO2(5μmol/L,10μmol/L,15μmol/L)were infected with LX-2 cells for 24h in a group design to construct the activation model of LX-2 induced by NaAsO2 in vitro,and a control group was set up.Mitochon-drial structure of LX-2 cells treated with NaAsO2 was observed by transmission electron microscopy(TEM).Fe2+levels were detected by fluorescence microscope and fluorescent enzyme label.The content of malondialdehyde(MDA)was determined by the colorimetric meth-od.The protein expression levels of SLC7A11,GPX4,and α-smooth muscle actin(α-SMA)were detected by Western blot.Results TEM showed that mitochondrial membrane integrity was damaged and mitochondrial ridges were reduced and disappeared in the NaAsO2group.In addition,compared with the control group,Fe2+levels in NaAsO2 treatment groups were increased(P＜0.05).There were statistically significant differences in MDA content among different doses of NaAsO2groups(F=7.18,P＜0.05).Compared with the control group,MDA content of LX-2 cells in 5 and 15μmol/L NaAsO2groups was higher than that in the control group(P＜0.05).At the translation level,the expression of fibrosis index α-SMA protein level was up-regulated with the increase of NaAsO2dose(P＜0.05),the protein expression levels of ferroptosis index SLC7A11 and GPX4decreased in a dose-dependent manner with the increase of the dose of NaAsO2(P＜0.05).Conclusion Ferroptosis is involved in the activation of LX-2 cells induced by NaAsO2.

Effects of sodium arsenite on mitochondrial function and expression of SIRT1/PGC-1α pathway-related proteins in human normal liver cell / 环境与职业医学

Background Long-term exposure to arsenic can cause liver injury of varying degrees. Mitochondrial damage may be an early key event of arsenic-induced liver injury. Silent mating type information regulation 2 homolog 1 (SIRT1)/ recombinant peroxisome proliferators-activated receptor gamma coactivator 1 alpha (PGC-1α) is an important pathway regulating mitochondrial mass and function. However, whether arsenic-induced liver injury is related to mitochondrial dysfunction mediated by SIRT1/PGC-1α pathway remains unclear. Objective To investigate potential effects of sodium arsenite (NaAsO2) on mitochondrial function and expressions of SIRT1/PGC-1α pathway-related proteins in human normal liver cell. Methods Human normal liver cells (MIHA cells) were used as the research object. MIHA cells were treated with different concentrations of NaAsO2 (0, 5, 10 and 20 μmol·L−1) for 24 h, and the cells were collected for study. The ultrastructure of mitochondria was observed by transmission electron microscopy, adenosine triphosphate (ATP) concentration by fluorescence method, mitochondrial membrane potential (MMP) level by flow cytometry, and SIRT1, PGC-1α and their downstream nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) protein expression levels by Western blotting. One-way analysis of variance and trend test were used for data statistical analysis. Results The viability of MIHA cells decreased gradually with the increase of NaAsO2 concentration (F=6495.47, P<0.001). The transmission electron microscope observation showed that the size of mitochondria in the 10 μmol·L−1 NaAsO2 treatment group was different, and the mitochondria were swollen or elongated in a rod-like shape. The mitochondria in the 20 μmol·L−1 NaAsO2 treatment group swelled like air spheres or vacuoles. The ATP concentration and MMP level of MIHA cells gradually decreased with the increase of NaAsO2 concentration (Ftrend of ATP=172.28, Ftrend of MMP=59.91, both Ps<0.001). Compared with the control group, the protein expression levels of SIRT1, PGC-1α, NRF1, and TFAM were not significantly changed in the 5 μmol·L−1 NaAsO2 treatment group, while the protein expression levels of SIRT1, PGC-1α, and TFAM were decreased in the 10 μmol·L−1 NaAsO2 treatment group, and the protein expression levels of SIRT1, PGC-1α, and NRF1 were decreased in the 20 μmol·L−1 NaAsO2 treatment group. The results of trend test showed that the protein expression levels of SIRT1, PGC-1α, NRF1, and TFAM decreased gradually with the increase of NaAsO2 concentration (Ftrend of SIRT1=47.07, P<0.001; Ftrend of PGC-1α=15.17, P<0.01; Ftrend of NRF1=13.54, P<0.01; F trend of TFAM=4.20, P<0.05). Conclusion The down-regulation of SIRT1/PGC-1α and its downstream NRF1 and TFAM may be involved in NaAsO2-induced mitochondrial dysfunction in liver cells.

Role of mitogen-inducible gene 6 in the activation of human hepatic stellate cells and deposition of extracellular matrix induced by sodium arsenite / 环境与职业医学

Background Arsenic is a well-known environmental toxicant. Hepatic fibrosis could occur dueto excessive or long-term exposure to arsenic, while associated molecular mechanisms remain undefined. Mitogen-inducible gene 6 (Mig-6) exhibits a protective effect on numerous diseases or cancers. However, the specific role of Mig-6 in the mechanisms of arsenite-induced hepatic fibrosis remains indistinct. Objective To investigate the specific role of Mig-6 in the activation of hepatic stellate cells (HSC) and the deposition of extracellular matrix (ECM) induced by sodium arsenite (NaAsO2). Methods Human hepatic stellate cells (Lx-2) were treated with 0, 1.875, 3.75, 7.5, and 15 μmol·L−1 of NaAsO2 for 24 h, or with 7.5 μmol·L−1 NaAsO2 for 0, 12, 24, 48, and 72 h. Additionally, Lx-2 cells were transfected by pcDNA3.1(+)/Mig-6, then treated with 7.5 μmol·L−1 NaAsO2 for 24 h; a blank control group, a pcDNA3.1(+)-control group, a pcDNA3.1(+)/Mig-6 group, and an arsenic (7.5 μmol·L−1 NaAsO2) group were also set up. After transfection, the cells and culture supernatants were collected, and the protein levels of Mig-6, α-smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1) in Lx-2 cells were identified by Western blotting analysis; moreover, the secretion levels of main ECM components in supernatants such as hyaluronic acid (HA), laminin (LN), collagens IV (COL-IV), and procollagen-III (PIIINP) were tested by ELISA. Results The Mig-6 expression decreased in the 3.75, 7.5, and 15 μmol·L−1 NaAsO2 groups (0.561±0.095, 0.695±0.048, and 0.401±0.030) compared to the control group (1.000±0.000) in Lx-2 cells (P<0.05). After administration with 7.5 μmol·L−1 of NaAsO2 for 24, 48, and 72 h, the Mig-6 expression (0.856±0.036, 0.515±0.077, 0.491±0.060) decreased compared with the 0 h group (1.000±0.000) (P<0.05). After over-expression of Mig-6, the results of Lx-2 activation related protein levels showed that compared to the control group, the α-SMA and TGF-β1 expression were up-regulated in the arsenic group (P<0.05); meanwhile, the α-SMA and TGF-β1 in the Mig-6 over-expression combined arsenic exposure group reduced compared to the arsenic (7.5 μmol·L−1) group (P<0.05). The results of ELISA showed that compared with the control group, the HA, LN, PIIINP, COL-IV in the arsenic group were up-regulated (P<0.05); while compared to the arsenic group, the HA, LN, PIIINP, and COL-IV in the Mig-6 over-expression combined with arsenic exposure group were decreased (P<0.05). Conclusion Arsenic down-regulates Mig-6 expression in HSC, and over-expression of Mig-6 can reverse the activation of HSC and ECM deposition induced by arsenic exposure. It suggests that Mig-6 plays a protective role in arsenic-induced HSC activation and ECM deposition.

Expressions and roles of key enzymes in serine synthesis pathway in NaAsO2-treated HaCaT cells / 环境与职业医学

Background The key enzymes of serine synthesis pathway (SSP) play an important role in tumor growth, proliferation, and invasion, but their roles in arsenic carcinogenesis are unclear. Objective To observe the effects of NaAsO2 treatment on the expressions of key enzymes [such as phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH)] of SSP and on the ability to proliferate and migrate in human immortalized skin keratinocytes (HaCaT) and NaAsO2-induced malignantly transformed HaCaT (T-HaCaT), and to explore the roles of SSP key enzymes in arsenic carcinogenesis. Methods (1) The T-HaCaT cells constructed earlier by our research team were divided into a passage control (0 μmol·L−1 NaAsO2) group, a T-HaCaT (0.5 μmol·L−1 NaAsO2) group, a NCT503 (PHGDH inhibitor, 25 μmol·L−1) group, and a NCT503 (25 μmol·L−1) + T-HaCaT (0.5 μmol·L−1 NaAsO2) group. Western blotting was used to detect the protein expression levels of SSP key enzymes in the passage control group and the T-HaCaT group. CCK8 assay and cell scratch test were used to detect the proliferation and migration rates of cells in each group respectively. (2) Well-grown logarithmic-phase HaCaT cells were treated with 0, 0.625, 1.25, and 2.5 μmol·L−1 NaAsO2 for 0, 24, 48, and 72 h to detect cell proliferation rate and protein expression levels of SSP key enzymes. In the subsequent experiment, HaCaT cells were pretreated with 25 μmol·L−1 NCT503 for 6 h, and then treated with 2.5 μmol·L−1 NaAsO2 for 72 h continuously. The experimental groups included a control (0 μmol·L−1 NaAsO2) group, an exposure (2.5 μmol·L−1 NaAsO2) group, a pretreatment (25 μmol·L−1 NCT503) group, and a pretreatment (25 μmol·L−1 NCT503) + exposure (2.5 μmol·L−1 NaAsO2) group, to detect the proliferation rate of cells in each group. Results The protein expression level of PHGDH in the T-HaCaT group were 1.60 times higher than that in the passage control group (P<0.05), and its proliferation rate (177.51%±14.69%) and migration rate (53.85%±0.94%) were also higher than the passage control group’s (100.00%±0.00% and 24.30%±2.26%) (both Ps<0.05), respectively. After the NCT503 intervention, the proliferation rate (144.97%±8.08%) and migration rate (35.80%±0.99%) of cells in the NCT503 + T-HaCaT group were lower than those in the T-HaCaT group (both P<0.05). The proliferation rate of HaCaT cells after NaAsO2 exposure for 72 h increased with the increase of exposure concentration (r=0.862, P<0.05), and consistently, the protein levels of SSP key enzymes in HaCaT cells in each exposure group were higher than those in the control group (all P<0.05). The proliferation rate of HaCaT cells treated with 2.5 μmol·L−1 NaAsO2 increased with the extension of exposure time (r=0.775, P<0.05), which was consistent with the changes of PHGDH levels in cells. After the NCT503 intervention, the proliferation rate of the pretreatment + exposure group was significantly lower than that of the exposure group (P<0.05). Conclusion The key enzymes of SSP may play an important role in the proliferation of T-HaCaT cells induced by NaAsO2.

Effect of sciadopitysin on sodium arsenite-induced senescence of rat hippocampal neurons / 环境与职业医学

Background In addition to the typical signs of skin damage, long-term arsenic exposure is often accompanied by signs and symptoms of neurobehavioral abnormalities. Objective To investigate potential intervention effect of sciadopitysin on senescence of neurons induced by sodium arsenite in rats and possible underlying mediating effect of cell cycle-related transcription factor E2F1. Methods SH-SY5Y cells were treated with 4 μmol·L−1 sodium arsenite for 24 h and intervened with 50 μg·mL−1 Ginkgo biloba extract (EGb761) or four major biflavonoids in Ginkgo biloba leaves (isoginkgetin, bilobetin, sciadopitysin, and ginkgetin) for 24 h respectively. Then, cell viability was measured by CCK-8 assay. Thirty-two 180-200 g SPF rats were randomly divided into a control group, an arsenic treatment group (10 mg·L−1), a Ginkgo biloba extract intervention group (10 mg·kg−1), and a sciadopitysin intervention group (10 mg·kg−1), 8 rats in each group, half male and half female. The rats were treated with sodium arsenite by free drinking water for 3 consecutive months, and the intervention treatment was conducted after 2 months of poisoning with drug intake by gavage for 1 month. HE staining was used to detect structural changes in the hippocampus, while Nissl's staining was used to detect changes in hippocampal morphology and neuron numbers. Moreover, senescence-associated β galactosidase (SA-β-gal) staining and Western blotting were used to detect senescence of hippocampal neurons and the expression level of E2F1, respectively. Results Compared to the arsenic treatment group, EGb761 and the four biflavonoids in Ginkgo biloba leaves effectively antagonized the inhibitory effect of sodium arsenite on cell viability (all Ps＜0.05), and sciadopitysin showed better restoration of cellular viability than Ginkgo biloba extract (P＜0.05). The results of HE staining and Nissl's staining showed that the hippocampal neurons in the arsenic treatment group were reduced in cell count and the synaptic structure was abnormal, with swelling, nuclear shrinkage, and vacuole, compared with the control group. The results of SA-β-gal staining showed that the number of senescent cells in the arsenic treatment group (15.75±3.01) was significantly increased compared with the control group (2.88±0.84) (P＜0.05); the numbers of senescent cells in the Ginkgo biloba extract group (9.38±1.92) and the sciadopitysin treatment group (7.75±2.38) were significantly decreased compared with the arsenic treatment group (all Ps＜0.05). The results of Western blotting showed that compared with the control group, the expression of E2F1 protein in hippocampus of the arsenic treatment group was significantly decreased (1.00±0.17 vs. 0.65±0.19, P<0.05); compared with the arsenic treatment group, the protein expression level of E2F1 in hippocampus of the sciadopitysin treatment group (0.89±0.18) was significantly recovered (P<0.05); compared with Ginkgo biloba extract (0.68±0.19), sciadopitysin had a better recovery effect on E2F1 expression level (0.89±0.18) (P＜0.05). The results of correlation analysis showed that the E2F1 protein expression level was negatively correlated with the positive rate of SA-β-gal staining in hippocampal neurons (r=−0.518, P＜0.05). Conclusion Sciadopitysin is an effective component of Ginkgo biloba extract. It can effectively inhibit the senescence of hippocampal neurons induced by sodium arsenite, and E2F1 may play an important mediating role.

Mechanism of BCL2L2-PABPN1 expression induced by sodium arsenite and its metabolites in 16HBE cells / 中国职业医学

@#Objective - - （BCL2L2）- （ ） To investigate the differential expression of the fusion gene BCL 2 like protein 2 poly A （PABPN1） （ ） binding protein nuclear 1 induced by sodium arsenite SA and its methylated metabolites in 16HBE cells and the Methods ） ， related mechanism. i The 16HBE cells exposed to SA at concentrations of 1.5 3.0 and 4.5 µmol/L were set as -， - - low medium and high dose arsenic exposure groups. The 16HBE cells exposed to 4.5 µmol/L monomethylarsonic acid （ ）， （ ） ， MMA dimethylarsonic acid DMA and SA were set as MMA group DMA group and SA group. The 16HBE cells without ， BCL2L2-PABPN1 toxic stimulation were set as control group. After the cells were cultured for 48 hours the expression of was - （ - ） ） （ ） detected by quantitative real time polymerase chain reaction qRT PCR . ii Two small interfering RNA siRNA silencing 基金项目：国家自然科学基金（ ）； 年云南省科技厅昆明医科大学应用基础研究联合专项面上项目 82160607 2021 （ ） 202101AY070001-054 作者简介：施雅（ —），女，在读大学本科生，主要从事劳动卫生与环境卫生学研究；尹锦瑶（ —），女，在读劳动卫生与环境卫 2001 1995 生学硕士研究生，主要从事劳动卫生与环境卫生学研究；施雅和尹锦瑶为共同第一作者 通讯作者：何越峰教授，博士研究生导师，- ： E mail heyuefeng@kmmu.edu.cn中国职业医学 年 月第 卷第 期 ， ， ， · · 2022 10 49 5 Chin Occup Med October 2022 Vol.49 No.5 523 BCL2L2-PABPN1， - fragments were designed and transfected into 16HBE cells to knockdown which were set as siRNA 1 group - - BCL2L2-PABPN1 and siRNA 2 group. Non transfected control group without knockdown of transfection was set up. After ， BCL2L2-PABPN1 - culturing for 48 hours the expression level of in the three groups of cells was detected by qRT PCR. The cell - survival rate and early apoptosis rate were detected by MTS method and JC 1 mitochondrial membrane potential detection ， （ ） ， method respectively. The apoptosis was detected by Hoechest33342/propidium iodide PI double staining and the expression - Results ） level of P53 signaling pathway related proteins was detected by Western blotting. i The relative expression of BCL2L2-PABPN1 （P ） BCL2L2- in 16HBE cells increased with the increasing SA doses <0.01 . The relative expression of PABPN1 - ， - - in high dose arsenic exposure was higher than that in control group low dose and medium dose arsenic exposure （ P ） BCL2L2-PABPN1 ， groups all <0.05 . The relative expression of in SA group was higher than those in control group MMA （ P ） BCL2L2-PABPN1 group and DMA group all <0.05 . The relative expression of showed no significant difference between ， （ P ） ） BCL2L2-PABPN1 control group MMA group and DMA group all >0.05 . ii The relative expression levels of and cell - - - （ P ） survival rate in siRNA 1 group and siRNA 2 group were lower than those in non transfected control group all <0.05 . ， （P ） However there was no significant difference in the early apoptosis rate among the three groups >0.05 . The results of - Hoechest33342/PI double staining showed that the number of nuclear shrinkage and early apoptotic cells in siRNA 1 group and - - ， - siRNA 2 group was higher than that in non transfected control group. The relative protein expression levels of P53 phospho ， - - ， - - （ P ） p53 BCL 2 associated death promoter P21 and cytochrome C in siRNA 1 group and siRNA 2 group were higher all <0.05 , - - P and the relative protein expression levels of P53 up regulated modulator of apoptosis were lower (all <0.05), when compared - Conclusion with the non transfected control group. SA may block the apoptosis of 16HBE cells by inducing the expression of BCL2L2-PABPN1 fusion gene . The mechanism may be related to the activation of P53 signaling pathway. The SA methylated BCL2L2-PABPN1 BCL2L2-PABPN1 - metabolites MMD and DMA had no effect on the expression of . may affect anti apoptosis BCL2L2 PABPN1 through affecting the synergistic effect of and genes.

Effects of chronic exposure to sodium arsenite on liver damage in rats / 预防医学

Objective@#To examine the effect of chronic exposure to sodium arsenite on liver damages in rats. @*Methods@#Fifty-six healthy adult SD rats (28 males and 28 females) were randomly divided into 4 groups. Rats in the low-, medium- and high-dose groups were given sodium arsenite solutions at doses of 2, 10 and 50 mg/L for successive 24 weeks, while animals in the control group were given deionized water. The rat body and liver weights were measured and the liver coefficient was estimated. The urine arsenic level was detected using atomic fluorescence spectrometry, and hepatic tissue sections were stained with uranium acetate and lead citrate for morphological observations under an electron microscope. @*Results@#The body weights of both male and female rats appeared a tendency towards a rise with the duration of exposure to sodium arsenite (male rat: Wald χ2=3 610.621, P<0.001; female rat: Wald χ2=2 186.217, P<0.001, and there were no significant differences in the rat body weight 24 weeks post-exposure to sodium arsenite in each group, while there was an interaction between time and group (male rat: Wald χ2=15.874, P=0.001; Wald χ2=9.460, P=0.024). There were significant differences in the rat liver weight and liver coefficient in each group (male rat: F=18.964 and 29.968, both P<0.001; female rat: F=11.919 and 15.070, both P<0.001), with the lowest liver weight (10.17±1.15) g and liver coefficient (1.99±0.21)% measured in male rats in the high-dose group, and the highest liver weight (12.91±1.29) g and liver coefficient (4.10±0.56)% in female rats in the high-dose group. The median urine arsenic levels (interquartile range) were 25.60 (30.27), 146.56 (101.06), 1 034.68 (600.06) and 3 796.98 (19 966.89) μg/L in rats in the control, low-dose, medium-dose and high-dose groups, respectively (χ2=50.211, P<0.001), and the urine arsenic level was significantly higher in the medium- and high-dose groups than in the control group (both P<0.001). Hepatic edema was seen in rats in the low- and medium-dose groups, and hepatic edema, focal hepatic cell necrosis, hyperplasia of bile capillaries and peri-bile capillary endolysis were observed in rats in the high-dose group.@*Conclusions@#Chronic exposure to arsenic may cause morphological alterations of rat hepatic tissues, and the rat hepatic damage aggravates with the dose of exposure to arsenic.

Effects of sodium arsenite on proliferation, apoptosis and Hippo-YAP pathway of PC12 cells / 中国药理学通报

To investigate the effects of sodium arsenite(NaAsO

Effect of dictyophora polysaccharide on mitophagy induced by sodium arsenite in L-02 cells / 中华地方病学杂志

Objective:To observe the effect of dictyophora polysaccharide (DIP) on PINK1/Parkin pathway mediated mitophagy induced by sodium arsenite (NaAsO 2) in human hepatocytes (L-02 cells). Methods:The L-02 cells in logarithmic growth phase and in good condition were divided into control group, NaAsO 2 group (10 μmol/L), DIP group (80 μg/ml), DIP + NaAsO 2 group (80 μg/ml DIP + 10 μmol/L NaAsO 2) , N-acetylcysteine (NAC) group (5 mmol/L), and NAC + NaAsO 2 group (5 mmol/L NAC + 10 μmol/L NaAsO 2). Western blotting was used to detect the expression levels of mitophagy related proteins p62, microtubule-associated protein 1 light chain 3 (LC3)Ⅱ/LC3Ⅰ, PINK1, and Parkin. The mitochondrial stucture and autophagosomes were observed by transmission electron microscope, the fluorescent probe method was used to detect the expression level of intracellular reactive oxygen species (ROS). Results:Compared with the control group, the protein expressions of p62, LC3 Ⅱ/LC3 Ⅰ, PINK1, and Parkin in NaAsO 2 group were higher ( P < 0.05); compared with the NaAsO 2 group, the protein expressions of p62, LC3 Ⅱ/LC3 Ⅰ, PINK1 and Parkin were lower in DIP, DIP + NaAsO 2, NAC, and NAC + NaAsO 2 groups ( P <0.05). According to the transmission electron microscope, compared with the control group, the mitochondria of L-02 cells in NaAsO 2 group were significantly damaged and the number of autophagosomes increased. Compared with NaAsO 2 group, the degree of mitochondrial swelling, vacuolar degeneration and the number of autophagosomes decreased in DIP + NaAsO 2 group. Compared with the control group (33 110.00 ± 2 191.28), the intracellular ROS level in NaAsO 2 group was higher (48 000.00 ± 2 395.31, P < 0.05); the level of intracellular ROS in DIP + NaAsO 2 group (38 670.00 ± 2 620.56) was significantly lower than that in NaAsO 2 group( P < 0.05), and there was no significant change compared with the control group ( P > 0.05). Conclusions:NaAsO 2 can induce PINK1/Parkin mediated mitophagy in L-02 cells. DIP can alleviate NaAsO 2 induced mitophagy. DIP may affect PINK1/Parkin mediated mitophagy induced by NaAsO 2 through the regulation of ROS.

Improvement Effects of α-lipoic Acid on Glucose Metabolism Disorder of Insulin Resistant HepG 2 Cells / 中国药房

OBJECTIVE：To study the i mprovement effects of α-lipoic acid on glucose metabolism disorder of insulin resistant HepG2 cells. METHODS ：The effects of 25-1 000 µmol/L α-lipoic acid on survival rate of human hepatoma cell HepG2 were determined by MTT assay so as to determine the concentration of α-lipoic acid. Negative control group ，insulin resistance group （1× 10－7 mol/L insulin ），combination resistance group （30 µmol/L sodium arsenite+ 1×10－8 mol/L insulin ），α-lipoic acid low- concentration，medium-concentration and high-concentration groups were set up. HepG 2 cells were treated with α-lipoic acid for 12 h and then cultured with corresponding concentration of sodium arsenite or/and insulin for 24 h. The glucose oxidase method was used to detect the glucose consumption ，colorimetric method was used to detect hexokinase activity and pyruvate kinase activity ， and anthrone method was used to detect glycogen content. Western blot assay was used to detect the protein expression of GLUT 4， p-GSK3β and GSK3β as well as the ratio of p-Akt/Akt and p-GSK3β/GSK3β. RESULTS：25，50，100 µmol/L α-lipoic acid had no significant effect on the survival rates of HepG 2 cells（P＞0.05），and survival rates of H epG2 cells were higher than 96％，so they were used as the low ，medium and high concentration for follow-up study. Compared with negative control group ，glucose consumption，the activities of hexokinase and pyruvate kinase ，glycogen content ，protein expression of GLUT 4 and p-GSK 3β，the ratio of p-Akt/Akt and p-GSK 3β/GSK3β were decreased significantly in insulin resistance group and combined resistance group， while the protein expression of GSK 3β was increased significantly（P＜0.05）. Compared with combination resistance group ，the glucose consumption （except for α-lipoic acid low- concentration group ），the activities of h exokinase（except for α-lipoic acid low-concentration and medium-concentration groups ） andpyruvate kinase （except for α-lipoic acid low-concentration com and medium-concentration groups ）， glycogen contents ， protein expression of GLUT 4 （except for α-lipoic acid mail：bliang163@163.com low-concentration group ）and p-GSK3β，the ratio of p-Akt/ Akt（except for α-lipoic acid low-concentration and medium-concentration groups ）and p-GSK 3β/GSK3β（except for α-lipoic acid low-concentration groups ）were increased significantly in α-lipoic acid groups ，while protein expression of GSK 3β（except for α-lipoic acid low-concentration and medium-concentration groups ） was decreased significantly （P＜0.05）；glycogen content ， protein expression of GLUT 4 and the ratio of p-GSK 3β/GSK3β in α-lipoic acid high-concentration group as well as the protein expression of p-GSK 3β in α-lipoic acid medium-concentration and high-concentration groups were improved significantly （P＜0.05）. CONCLUSIONS：α-lipoic acid can improve the disorder of glucose metabolism in insulin resistant HepG 2 cells，the mechanism of which may be associated with the increase of glucose consumption ，the activities of glucose metabolism related enzymes and glycogen content ，and expression up-regulation of the phosphorylation levels of Akt and GSK 3β protein，the expression of GLUT 4 and p-GSK 3β proteins，down-regulation of the expression of GSK 3β protein.

Effects of arsenite and MPST over-expressin on GRP78/CHOP endoplasmic reticulum stress pathway in SH-SY5Y cells / 中国药理学通报

Aim To investigate whether endoplasmic reticulum stress is involved in the neurotoxicity of sodi¬um arsenite and clarify whether over-expression of 3-mercaptopyruvate sulfurtransferase (MPST) regulates endoplasmic reticulum stress induced by arsenic. Methods The SH-SY5Y cell line stably expressing the exogenous MPST gene was obtained by constructing the lentiviral vector of MPST gene. The SH-SY5Y cells were randomly divided into six groups, the SR-MPST over-expression group stably expressing the exogenous MPST gene, SH-PEB control group transfected with empty vector, the arsenite treatment group ( NaAs02 group ), TUDC A treatment group ( blocker of endoplasmic reticulum stress ) and TUDC A + NaAs02 group. Western blot was used to examine the protein expression of GRP78 and CHOP after different treatment. Results Although MPST overexpression had no significant effects on the expression of GRP78 and CHOP proteins, NaAs02 could significantly increased the protein levels of GRP78 and CHOP ( P < 0. 01 ) and the up-regulation of GRP78 and CHOP proteins caused by NaAs02 could be blocked by the treatment of TUDC A. In addition, the inhibition by MPST overexpression on the arsenic-induced increase of GRP78 and CHOP proteins (P <0. 01 ) could also be reversed by the TUDC A treatment significantly. Conclusions The GRP78/ CHOP endoplasmic reticulum stress pathway is involved in the neurotoxic damage induced by arsenic; MPST overexpression may decrease arsenic-induced endoplasmic reticulum stress.

Effects of Curcumin on Sodium Arsenite Induced Neoplastic Cell Transformation in Balb/c 3T3 Cells

HIGHLIGHTS Sodium arsenite can cause neoplastic transformation in cells. Curcumin reduced cell viability and increased LDH activity in transformed Balb/c 3T3 cells. Curcumin caused DNA damage in transformed Balb/c 3T3 cells. Curcumin may play a protective role in sodium arsenite-induced toxicity.

Abstract Arsenic is a toxic substance that spreads widely around the environment and accumulates as metalloid in the earth's crust. Arsenic and its derivatives are found in drinking water, nutrients, soil, and air. Exposure to arsenic is associated with lung, blood, skin cancer and various lesions. Curcumin is a polyphenolic compound derived from Curcuma longa (turmeric) rhizome and is one of the main curcuminoids. Curcumin is known to be antioxidant, antibacterial, anti-inflammatory, analgesic effects. This study aimed to investigate the potential of sodium arsenite to transform embryonic fibroblast cells and to evaluate the cytotoxic and genotoxic effects of curcumin in neoplastic transformed cells. Neoplastic cells transformation was induced by sodium arsenite in Balb/c 3T3 cells at the end of 32 days. After transformation assay, the transformed cells were treated with various concentration of curcumin to evaluate cell viability, lactate dehydrogenase activity and DNA damage for 24h. The results revealed that curcumin decreased cell viability and increased the activity of lactate dehydrogenase enzyme in neoplastic transformed Balb/c 3T3 cells. In conclusion, the results demonstrated that curcumin has an anticancer effect on neoplastic transformed Balb/c 3T3 cells by causing DNA damage.

Effect of sodium arsenite on cell cycle of SH-SY5Y cells and intervention of MPST over-expression / 中国药理学通报

Aim To investigate the effects of sodium arsenite (NaAsO2) on the cell cycle and growth, and the intervention of MPST over-expression in the neural cells. Methods NaAsO2 was used to treat SH-SY5Y neuroblastoma cells for 48 h from the blank control (BC), empty vector control ( transfected with empty vector, NC ) and over-expression group ( lentiviral transfection with MPST,OP). The methods of CCK-8, crystal violet staining,flow cytometry and Western blot were used to examine cell viability,adherent rate,cell cycle and protein expression of p53,CDC25A,CyclinA and CDK2. Results The cell viability and adherent rate significantly decreased after treatment with NaAsO2 for 48 h,which was reversed in OP group (P <0. 01). Meanwhile, NaAsO2 also significantly increased the proportion of S phase cells and p53 protein expression, and down-regulated the protein levels of CDC25A,Cyc-lin A and CDK2 in BC and NC groups ( P < 0. 01), whereas the above changes of protein levels were significantly antagonized in OP group compared with NC group (P < 0. 05, P < 0. 01). Conclusions NaAsO2 inhibits the cell growth by inducing S-phase arrest and over-expression of MPST could reverse the noxious effects caused by NaAsO2 in SH-SY5Y cells.

The establishment of the immortalized mouse brain microvascular pericytes model and its preliminary application in screening of cerebrovascular toxicants / 中华预防医学杂志

Objective@#To establish the immortalized mouse brain microvascular pericytes model and to apply to the cerebrovascular toxicants screening study.@*Methods@#Brain pericytes were isolated from 3 weeks of mice by tissue digestion. Immortalized pericyte cell line was constructed by infecting with LT retrovirus. Monoclone was selected to purify the immortalized pericyte cell line. The pericyte characteristics and purity were explored by immunocytochemistry. Cell proliferation was measured by using the Pomega MTS cell Proliferation Colorimetric Assay Kit. Pericytes were treated with 0, 160, 320, 640, 1 280, 2 560 μmol/L lead acetate, 0, 5, 10, 20, 40, 80 μmol/L cadmium chloride and 0, 5, 10, 20, 40, 80 μmol/L sodium arsenite in 24 hours. Cell toxicity of each group was determined by MTS assay, median lethal dose (LD50) was calculated in linear regression.@*Results@#Mouse brain pericytes were successfully isolated by tissue separation and enzyme digestion method. After immortalized by LT retroviruses, monoclone was selected and expanded to establish pericyte cell line. The brain pericytes exhibited typical long spindle morphology and positive staining for α-SMA and Vimentin. The proliferation of brain pericytes cell lines was very slowly, and the doubling time was about 48 hours. The proliferation of immortalized brain pericytes cell lines was very quickly, and the doubling time was about 24 hours. After lead acetate, cadmium chloride and sodium arsenite treatment for 24 hours respectively, gradual declines in cell viability were observed. The LD50 of lead acetate was 2 025.0 μmol/L, the LD50 of cadmium chloride was 36.6 μmol/L, and the LD50 of sodium arsenite was 33.2 μmol/L.@*Conclusion@#The immortalized mouse brain microvascular pericyte model is established successfully by infecting with LT retrovirus, and can be applied to screen cerebrovascular toxicants. The toxicity of these toxicants to immortalized mouse brain microvascular pericyte is in sequence: sodium arsenite,cadmium chloride, lead acetate.

Effects of sodium arsenite exposure on activation and extracellular matrix secretion of human hepatic stellate cells / 中华预防医学杂志

Objective@#To explore the effects of sodium arsenite (NaAsO2) exposure on the activation and extracellular matrix secretion of human hepatic stellate cells, and to provide a theoretical basis for the mechanism study of arsenic induced hepatic fibrosis.@*Methods@#Different doses of NaAsO2 (0.0, 0.1, 1.0, 10.0, 50.0, 100.0 μmol/L) were exposed to human hepatic stellate cell line (Lx-2) for 24, 48 and 72 huors. CCK-8 assay was used to measure cell viability and IC50 of NaAsO2 on Lx-2 was then calculated; According to IC50 results, 0.000, 1.875, 3.750, 7.500, and 15.000 μmol/L of NaAsO2 were exposed to Lx-2 cells for 24 hours, besides, 7.500 μmol/L of NaAsO2 was exposed to Lx-2 cells for 0, 12, 24, 48, and 72 hours, then collected cells and culture supernatant; HSC activation-related protein, including α-smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1) expression levels were detected by Western blot analysis, the main extracellular matrix including laminin (LN) , hyaluronic acid (HA), collagen Ⅳ (COL-Ⅳ) and procollagen Ⅲ(P Ⅲ NP) secretion level was detected by Elisa assay.@*Results@#CCK-8 assay showed that the cell viability of Lx-2 cells were increased obviously at low doses (≤1.0 μmol/L) of arsenic exposure, especially at 48 and 72 h. In contrast, with the increasing doses of arsenic exposure, the survival rate of Lx-2 cell was decreased gradually, and the survival rate of the high-dose (50, 100 μmol/L) arsenic exposure group at 24, 48 and 72 h were significantly lower than 0.0 μmol/L group, P<0.05. The IC50 of NaAsO2 on Lx-2 cells at 24, 48, 72 h were calculated as 72.75, 48.19 and 29.95 μmol/L, respectively; The expression levels of HSC activation-related protein showed that, after treated with 1.875, 3.750, 7.500, 15.000 μmol/L NaAsO2 for 24 h, α-SMA and TGF-β1 protein level were higher than 0.000 μmol/L group. The increased expression of α-SMA and TGF-β1 protein were most significant in 7.500 μmol/L NaAsO2 group (P<0.05). In addition, the expression levels of α-SMA and TGF-β1 also showed a time-dependent increasing in Lx-2 cells after treated with 7.500 μmol/L NaAsO2 for 0, 12, 24, 48 and 72 h; Elisa assay showed that after treated with 1.875, 3.750, 7.500, 15.000 μmol/L NaAsO2 for 24 h, the secretion levels of HA, LN, COL-Ⅳ and PⅢNP were obvious higher than 0.000 μmol/L group (P<0.05). Moreover, the secretion levels of HA, LN, COL-Ⅳ and P Ⅲ NP also showed a time-dependent increased manner in Lx-2 cells after exposed to 7.500 μmol/L NaAsO2 for 0, 12, 24, 48 and 72 h (P<0.05).@*Conclusion@#NaAsO2 exposure to Lx-2 cells can upregulate the expression level of HSC activation-related proteins, induce its further activation, then increase ECM secretion level.

Ameliorating effects of Raphanus sativus leaves on sodium arsenite-induced perturbation of blood indices in Swiss albino mice

Objective: To evaluate the ameliorating effects of Raphanus sativus leaves (RSL) against sodium arsenite(Sa)-induced adverse effects through mice experiments. Methods: Swiss albino mice were divided into four equal groups: control, Sa, RSL, RSL + Sa. Sa (10 mg/kg body weight/day), and powder form of RSL (50 mg/kg body weight/day) were provided as food supplement orallty. Blood indices were measured using commercially available kits through colorimetric methods. Results: It was observed that lactate dehydrogenase, alkaline phosphatase, alanine aminotransferase,and aspartate aminotransferase activities were significantly(P<0.05) higher in Sa-treated mice than those in the control group.RSL significantly reduced Sa-induced elevation of the activities of these enzymes in serum significantly (P < 0.05). Serum butyrylcholinesterase activity and high density lipoproteins cholesterol levels in Sa-treated mice were significantly (P < 0.05) lower than the control group, and the food supplementation of RSL could significantly(P<0.05)prevent the reduction of Sa-mediated serum butyryl cholinesterase activity and high density lipoproteins cholesterol levels.RSL could also reduce the Sa-induced elevation of serum urea level significantly(P<0.05). Conclusions: Results of this study suggest the protective or ameliorating effects of RSL on Sa-induced perturbation of blood indices are related to the hepatic,cardiovascular and kidney dysfunction.Therefore,RSL may be useful to reduce arsenic toxicity in human in the future.

Mechanism of Sodium arsenite by Wnt signaling pathway inhibits the proliferation of oral squamous cell carcinoma and promote apoptosis / 中国生化药物杂志

Objective To investigate the effect of sodium arsenite by Wnt signaling pathway on proliferation and apoptosis of oral squamous cell carcinoma.Methods Cell proliferation was detected after 1.25,2.5,5,10,20μmol/L sodium arsenite treatment human oral squamous cell carcinoma cell line Tca8113 for 24,48,72 hours by CCK8 experiment.0 and 14μmol/L sodium arsenite was used to treatment Tca8113 cells with 48h,cell apoptosis were detected by flow cytometry,Cleaved Caspase3,β-catenin,Cyclin D1 protein expression were detected by Western blot.Tca8113 cells were divided into control group,sodium arsenite group,activating agent+sodium arsenite group,all treated for 48hour,cell proliferation,apoptosis and Cleaved Caspase3,β-catenin,Cyclin D1 protein expression were detected by CCK8 assay,flow cytometry and Western blot.Results Tca8113 cell proliferation was inhibited significantly with the increase of treatment time and sodium arsenite concentration,and has a time and concentration dependent manner(P<0.05 or P<0.01).10μmol/L sodium arsenite as a follow-up study according to the IC50.Cell inhibition rate,apoptosis rate and Cleaved Caspase3 protein expression in 10μmol/L group were significantly higher than that of 0 mol/L group,the expression of β-catenin,Cyclin D1 protein was significantly lower than that of 0 mol/L group(P<0.01).Apoptosis rate,cell inhibition rate and Cleaved Caspase 3 protein expression in sodium arsenite group and activating agent+sodium arsenite group were significantly higher than control group,the expression of β-catenin and Cyclin D1 protein were significantly lower than control group(P<0.01).Apoptosis rate,cell inhibition rate and Cleaved Caspase 3 protein expression in activating agent + sodium arsenite group were significantly lower than that of sodium arsenite group,the expression of β-catenin and Cyclin D1 protein were significantly higher than that of sodium arsenite group(P<0.01).Conclusion Sodium arsenite can inhibit the proliferation of oral squamous cell carcinoma and promote apoptosis,and the mechanism was related to regulation of Wnt signaling pathway.

The effects of sodium arsenite on mRNA expression level of pigment epithelium-derived factor and apoptosis-related factors in PC12 cells / 中华地方病学杂志

Objective To observe the effects of different levels of sodium arsenite ( NaAsO2) on mRNA expression of pigment epithelium-derived factor (PEDF) and apoptosis-related factors in PC12 cells ( rat neuron properties pheochromocytoma). Methods PC12 cells were treated with different levels of NaAsO2 [0 (control group), 2, 5, 10 μmol/L] for 24 hours. The mRNA expression of PEDF and apoptosis-related factors (Bax, Bcl-2) were detected by real-time quantitative PCR. Results There were significant differences in the mRNA expressions of PEDF between the 4 groups (F=102.28, P 0.05); there were significant differences in the mRNA expressions of Bcl-2 between the 4 groups (F=19.87, P<0.05), the mRNA expressions of Bcl-2 in the group of 2, 5, 10 μmol/L (0.65 ± 0.03, 0.49 ± 0.04, 0.57 ± 0.09) were lower than that of control group (0.95 ± 0.11, all P<0.05);there were significant differences in the mRNA expressions of Bax/Bcl-2 between the 4 groups (F=8.352, P<0.05), the mRNA expressions of in the group of 5, 10μmol/L (1.80 ± 0.72, 1.82 ± 0.36) were higher than that of control group (1.02 ± 0.24, all P<0.05). Conclusion NaAsO2 may increase the expression of apoptosis-related factorsBax/Bcl-2 mRNA by decreasing the expression of PEDF mRNA in PC12 cells, leading to apoptosis in PC12 cells.