| Purpose: By establishing experimental model in vitro/in vivo of arsenic-induced abnormal skin keratosis, the study aims to know the distribution, metabolic characteristics and laws of arsenic in rabbits skin tissues and Haca T cells, and the influence of arsenic on skin cells’ proliferation and apoptosis, and skin damage. After studying, the paper is to explore the function of Nrf2-ARE signaling pathway in arsenic-induced skin keratosis and the oxidative damage mechanism in arsenic skin toxicity; After studying differential proteins in arsenic-induced abnormal skin keratosis, it is to discuss mechanisms in skin damage induced by arsenic at the level of protein molecules and to seek sensitive markers, so as to offer new thinking and scientific theory basis for prevention and treatment of arsenic-induced skin damage. Method: 1) MTT reduction method was adopted to test the growth of Haca T cells, to determine LC50 and exposure concentrations; exposure concentrations: 1/50, 1/20, 1/10 of 24 h LC50 respectively, that is, 1.30μmol/L, 3.25μmol/L and 6.50μmol/L; observation time: 24 h, 48 h and 72h; 2) Flow cytometry was used to test the apoptosis of Haca T cells; 3) 30 healthy clean-level male adult rabbits were randomly divided into 5 groups, 6 rabbits every group, consisting of the control group(deionized water) and sodium arsenite exposed groups. They were exposed for 12 weeks by means of free drink with the exposure doses of 1/100, 1/50, 1/20 and 1/10 of LD50, that is, L: 0.13mg/(kg.w), M: 0.26mg/(kg.w), MH: 0.65mg/(kg.w) and H: 1.30mg/(kg.w); 4) After the end of 12-week exposure, skin at the same position of rabbits were taken to observe damage of rabbits’ skin under electron microscope; 5) 24 h urine samples before executed were collected, and liver tissues were taken. Contents of i AsⅢ, i AsⅤ, DMA and MMA in urine, skin and liver of rabbits were detect by HPLC-HGAFS method; 6) Expression level of Nrf2 m RNA in Haca T cells and rabbits’ skin were test through PCR(Real-time polymerase chain reaction, RT-PCR); 7) The contents or vitality of CAT, 8-OHd G, MPO, GSH-Px, GST, SOD, MDA and HO-1 in rabbits’ skin were tested using relevant kits, and the total protein was tested using BCA method; 8) The differently expressed protein spots of arsenic-induced rabbits’ keratotic skin were screened using two-dimensional gel electrophoresis(2-DE), and differential proteins were identified through in-gel digestion, matrix-assisted laser desorption/ ionization time of flight mass spectrometry(MALDI-TOF-MS) and NCBI(nr) non-redundant database retrieve; 9) The expression level of CK1 and CK10 m RNA in Haca T cells and CK1, CK10, CK2 and protein disulfide isomerase(PDI) m RNA in rabbits’ skin were test using RT-PCR. Results: 1) 1.30μmol/L sodium arsenite exposure for 48 h can promote Haca T cell proliferation remarkably; 3.25μmol/L sodium arsenite exposure for 96 h and 6.25μmol/L sodium arsenite exposure for 72 h can inhibit Haca T cell proliferation remarkably(P<0.05); 2) 1.30μmol/L sodium arsenite exposure can inhibit apoptosis of Haca T cell with the lowest apoptosis rate at 24h; 3.25μmol/L and 6.25μmol/L sodium arsenite exposure for 48 h and 72 h may gradually increase apoptosis of Haca T cell(P<0.05); 3) Skin epidermis, especially structure of keratoderma and skin cellular morphology of rabbits has taken on great changes, that is, increased keratosis and abnormal keratosis under electron microscope. Compared with control group, 0.13mg/(kg.w) sodium arsenite exposure may cause the occurrence of abnormal keratosis, and the damage degree of rabbit keratosis was aggravated with the increase in the dose of sodium arsenite exposure; 4) After exposure for 12 weeks, the total arsenic, i AsⅢ and DMA level in rabbits’ urine took on the trend of gradual increase with the increase of exposure dose. The arsenic exposure group with H dose had higher content level compared with control group(P<0.05), and the i AsⅤcontent level had no obvious change trend. The MMA content level took on the trend of gradual increase with the increase of exposure dose and the arsenic exposure groups with MH and H dose had higher MMA content level compared with control group(P<0.05). All exposure groups had higher PMI compared with control group(P<0.05). Except the arsenic exposure groups with L dose, other exposure groups had higher SMI compared with control group(P<0.05). The total arsenic and DMA contents in rabbits’ skin of all exposure groups were higher than that of the control group(P<0.05), and those contents were increased with the increase of exposure dose. In all forms of arsenic, the DMA content was the highest. Except MH group, the contents of i AsⅢin all exposure groups were higher than that in the control group(P<0.05); the content of i AsⅤ in all exposure groups were higher than that in the control group; the MMA contents in L group and H group were lower than that in the control group while MMA content in MH group was higher than that in the control group(P<0.05). The total arsenic content level in rabbits’ liver took on the trend of gradual increase with the increase of exposure dose, and the total arsenic content level in M, MH and H groups were higher compared with the control group with significant difference(P<0.05). The i AsⅢcontent in liver had no significant difference in exposure groups with difference doses; the i AsⅤcontent in liver in M group was higher than other groups(P<0.05). The MMA content in liver had no significant difference in exposure groups with difference doses; The DMA contents in liver were different in exposure groups with difference doses, and the DMA contents in liver in MH and H groups were increased compared with the control group(P<0.05). The DMA arsenic form was the main form in rabbits’ skin and urine, and no law was found in various arsenic forms in liver. DMA proportion in rabbits skin tissue took on increasing trend with the increase of exposure dose. The total arsenic content and DMA content in rabbits skin and urine was in positive correlation with those in liver; 5) Sodium arsenite exposure could change Nrf2 m RNA expression in Haca T cells, and 1.30μmol/L sodium arsenite exposure could up-regulate m RNA expression. With the extension of exposure time and the increase of exposure concentration, m RNA expression was down-regulated(P<0.05). Expression level of Nrf2 m RNA was in positive correlation with cell viability in Haca T cells with arsenic exposure for 48h; Nrf2 m RNA expression in rabbits’ skin in L and M groups was up-regulated while Nrf2 m RNA expression in MH and H groups was down-regulated, wherein, the difference of L and H groups had statistical significance(P<0.05). 6) The contents of 8-OHd G in H dose of arsenic exposure groups and MDA in MH and H dose of arsenic exposure groups were increased compared with control group in rabbits skin(P<0.05); The vitality of CAT and HO-1 in M, MH and H dose of arsenic exposure groups were reduced compared with control group in rabbits skin(P<0.05); The vitality of GSH-Px in H dose of arsenic exposure groups were reduced compared with control group in rabbits skin(P<0.05); The vitality of GST in H dose of arsenic exposure groups were increased compared with control group in rabbits skin(P<0.05); The vitality of SOD and MPO had no significant difference in all groups; Expression level of Nrf2 m RNA was in positive correlation with the vitality of HO-1and GSH-Px while was negative correlation with the contents of 8-OHd G and MDA in rabbits skin; 7) 2-DE electrophoresis protein spots were apparently visible, with the number ranging from 100 to 200, wherein, the number of protein spots in control group was 119 while the number of protein spots in the left 4 groups was 129 in each group in full match. The match rate of protein spots in control group and left groups was 92.24%. There were 45 protein spots with different expression compared from the control group, and 20 differential proteins were identified. Among them, cytokeratin type Ⅱ, PDI and GST-P may be related to the toxicity of arsenic; 8) Cytokeratin type Ⅱhad expression in rabbits’ skin in groups with keratosis, while had no expression in the control group; Sodium arsenite exposure make Haca T cells CK1, CK10 m RNA expression changes, 1.30μmol/L dose enables upregulated m RNA expression, exposure increase while exposure time and dose increased gradually lowered m RNA expression(P<0.05). CK1, CK2 and CK10 m RNA in rabbits skin was up-regulated in L dose of arsenite exposure groups while reduced to the normal or down-regulated in H dose of arsenite exposure groups(P<0.05). PDI protein expression in rabbits skin was down-regulated first and up-regulated next with the increase of arsenite exposure dose; PDI m RNA expression in MH and H groups was un-regulated with the increase of arsenite exposure dosein in rabbits skin(P<0.05). Conclusion: Sodium arsenite exposure can be load and metabolism in Skin tissue and keratinocytes and induced oxidative damage in skin. It caused skin tissue and keratinocytes Nrf2 change, this change varies with the exposure concentration and time was bidirectional, Nrf2 change then affected keratinocytes proliferation and apoptosis while impacted on the vitality of downstream enzymes of Nrf2 signaling pathway in skin tissue, oxidation and antioxidation balance in skin was destroyed, disorder of keratinization was occured. PDI and CK1, CK2 and CK10 may play a role in the skin disorders induced by arsenic. And the change of Cytokeratin was a sensitive indicator... |
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