The Changes Of LncRNA And M~6A Modification In Pancreatic β Cell Damage Induced By Cadmium And The Effect Of Water-soluble Extract Of Bombyx Batryticatus On Cadmium Toxicity

Objective1、In order to clarify the change and correlation of m⁶A and its modified multiple LncRNAs in cadmium sulfate（CdSO₄）induced NIT-1 cell damage in pancreaticβ-cells.2、To explore the effect of the Water-soluble Extracts of the Beauveria Bassiana（BBWE）on the NIT-1 cell damage induced by CdSO₄.Methods1、Construction of CdSO₄-induced NIT-1 cells damage:CCK-8 assay,Hoechst staining,fluorescent probe method and oxidative damage index assay were used,to observe the magnitude of cell survival after staining NIT-1 cells by various concentrations of CdSO₄（0,1μM,2μM,4μM,8μM,16μM,32μM and 64μM）for 24 h,the apoptosis level,ROS content,insulin level,malondialdehyde（MDA）content and superoxide dismutase（SOD）activity were measured after（0,1μM,2μM and 4μM）CdSO₄treatment of cells for 24 h.2、To clarify the changes of m⁶A modification and its related enzymes and LncRNAs in CdSO₄-induced NIT-1 cell damage:RT-PCR was used to detect methyltransferase-like 3（METTL3）in m⁶A methyltransferase,fat mass and obesity-associated protein（FTO）in m6A demethylase,YTH protein family members in m6A methyl-binding protein（YTHDF1,YTHDF2,YTHDC1）in m RNA levels and LncRNAs（LncRNA-MALAT1,LncRNA-PVT1,LncRNA-XIST,LncRNA-TUG1 and LncRNA-NEA1）.Protein immunoblotting assay to detect the protein expression levels of methyltransferase-like 3（METTL3）in m⁶A methyltransferase,fat mass and obesity-associated protein（FTO）in m6A demethylase,and YTH protein family members（YTHDF1,YTHDF2,YTHDC1）in m⁶A methyl-binding protein.The level of m⁶A modification on total RNA was detected by Epi Quick TM RNA methylationquantification kit.3、To explore the effect of BBWE on NIT-1 cell damage caused by CdSO₄:cold water soaking method was used to extract water-soluble components of Beauveria bassiana,according to the CCK-8 assay,Hoechst staining and fluorescent probe method and the related oxidative damage kit,the cell survival rate of BBWE（0,0.4mg/m L,0.8mg/m L,1.6mg/m L,3.2mg/m L and 6.4mg/m L）stained NIT-1 cells for 24h was detected first,then NIT-1 cells were co-stained with 0.4 mg/m L and 0.8 mg/m L BBWE and 4μmol/L CdSO₄,respectively,to detect apoptosis level,ROS level,protein level,SOD activity and MDA content,and to study the effect of BBWE on CdSO₄-induced damage in NIT-1 cells.Results1、CdSO₄can induce NIT-1 cell damage:（1）Cell survival rate:Under the microscope,after cells treated with different concentrations of CdSO₄（0,1μM,2μM,4μM,8μM,16μM,32μM and 64μM）for 24 h,with the increase of CdSO₄concentration,the cells gradually shrunk,rounded and their ability to adhere to the wall was weakened.The results of CCK-8experiments showed that the cell survival rates of each treatment group were 100.00%,90.66%,78.06%,70.76%,57.43%,45.23%,32.72%and 23.21%,respectively.Compared with the control group,the differences in each treatment group were statistically significant（P<0.05）.（2）Apoptosis rate:It could be observed under fluorescence microscope that the apoptotic cells in the control group were less and the color of the nucleus was uniform blue staining,the apoptosis of the other CdSO₄treatment groups（1μM,2μM,4μM）gradually increased,and the nucleus was densely stained or fragmented and densely stained,turning white and shiny,showing a typical apoptotic morphology.The quantitative results showed that the apoptosis rate of the control group was 3.47%,while the apoptotic cells in the CdSO₄-treated group were 7.03%,17.23%and 34.64%,respectively（P<0.05）.（3）Cell oxidative damage:It can be observed under a fluorescence microscope that with the increase of CdSO₄concentration,the level of intracellular ROS gradually increases,Quantitative analysis revealed that the ROS generation in the cells of the 1μM CdSO₄-treated group was1.13 times that in the control group,and the ROS generation in the cells of the 2μM and 4μM CdSO₄-treated groups was 1.23 and 1.53 times that in the control group,respectively,with statistically significant differences compared with the control group（P<0.05）.In addition,the insulin level and SOD activity of the cells in the 1μM CdSO₄group tended to decrease and the MDA content tended to increase,but none of them was statistically significant compared with the control group.In contrast,the insulin levels（3.41 m IU/L,2.84 m IU/L）and SOD activities（13.59 U/mg protein,10.95 U/mg protein）in the cells of the 2μM and 4μMCdSO₄groups,compared with the control group（6.65 m IU/L,16.48 U/mg protein）were reduced（P<0.05）,The MDA content of cells in the 2μM and 4μM CdSO₄groups（15.44nmol/mg protein,22.47 nmol/mg protein）was increased compared with the control group（8.94 nmol/mg protein）（P<0.05）.2、To clarify the changes of m⁶A modification and its related enzymes and LncRNAs in CdSO₄-induced NIT-1 cell damage:（1）m⁶A-related enzyme levels:the m RNA levels of METTL3 in the 2μM and 4μM CdSO₄groups were 0.67 and 0.36 times that in the control group,respectively（P<0.05）,and the m RNA levels of FTO were 0.77 and 0.47 times that in the control group（P<0.05）.METTL3 protein levels in the CdSO₄group（1μM,2μM and 4μM）were 0.81,0.52 and 0.33 times higher than those in the control group,and FTO protein levels were 0.88,0.64 and 0.50 times higher than those in the control group,respectively.And the intracellular YTHDF1 m RNA level and protein expression,YTHDF2 m RNA level and protein expression,and YTHDC1 m RNA level and protein expression in each CdSO₄concentration group were not statistically different compared with the control group.（2）Levels of LncRNAs:compared with the control group,the levels of LncRNA-MALAT1 in cells of（1μM,2μM and 4μM）CdSO₄group were 0.84,0.70 and 0.36 times higher than those of the control group（P<0.05）,and the levels of LncRNA-PVT1 were 0.85,0.81 and0.44 times higher than those of the control group（P<0.05）,while the levels of LncRNA-TUG1,LncRNA-XIST and LncRNA-NEAT1 in the cells of the CdSO₄-exposed group were not statistically different compared with the control group;（3）Correlation between m⁶A and LncRNA levels:the m⁶A modification levels on total intracellular RNA in the（2μM,4μM）CdSO₄-treated group were significantly lower than those in the untreated group（P<0.05）,and the reduced m⁶A was positively correlated with LncRNA-PVT1 and LncRNA-MALAT1 levels,with correlation coefficients of 0.8200 and 0.9152,respectively.3、Effect of BBWE on cadmium-induced NIT-1 cell injury:（1）Cell survival and apoptosis:after（0,0.4 mg/m L,0.8 mg/m L,1.6 mg/m L,3.2 mg/m L and 6.4 mg/m L）BBWE was exposed to NIT-1 cells for 24 h,the cell survival rates were 100%,127.61%,153.07%,162.40%,140.78%and 42.23%,respectively,and the cell survival rate showed a trend of increasing and then decreasing with the increase of BBWE concentration compared with the control group（P<0.05）.During the co-treatment,the cell survival rate of（0.4mg/m L and0.8mg/m L）BBWE group was 130.06%and 156.00%,respectively,and the cell survival rate of 4μM CdSO₄was 52.19%,After BBWE（0.4mg/m L and 0.8mg/m L）was co-treated with4μM CdSO₄,the cell survival rates were 38.43%and 31.70%（P<0.05）.Under pretreatment conditions,the cell survival rates of（0.4mg/m L and 0.8mg/m L）BBWE were 106.09%and116.15%,respectively,the cell survival rates of 4μM CdSO₄were 60.98%,（0.4mg/m L and0.8mg/m L）BBWE was co-treated with 4μM CdSO₄,and the cell survival rates were 54.37%and 51.39%（P<0.05）.In addition,the cells in the combined 4μM CdSO₄and 0.8 mg/m L BBWE treatment groups were rounded,the nuclei were densely and densely stained,and the apoptosis rates were 5.06 and 1.31 times higher than those in the 0.8 mg/m L BBWE and 4μM CdSO₄groups,respectively（P<0.05）.（2）Cellular ROS levels:Under co-treatment,it was observed microscopically that cellular reactive oxygen species ROS were less in the control and 0.8 mg/m L BBWE groups,while ROS were significantly increased in the 0.8 mg/m L BBWE+4μM CdSO₄group,which were 1.18 and 1.05 times higher than those in the BBWE and CdSO₄groups,respectively（P<0.05）.（3）Oxidative damage related indexes:the results of BCA protein content measurement showed that the cellular protein content of 0.8 mg/m L BBWE,4μM CdSO₄and 0.8 mg/m L BBWE+4μM CdSO₄groups were 0.86,0.67 and 0.79times higher than that of the control group,respectively（P<0.05）,the cellular protein content in the CdSO₄group was 0.78 times higher than that in the BBWE group（P<0.05）.In the 4μM CdSO₄group and the 0.8 mg/m L BBWE with 4μM CdSO₄co-treatment group,the cellular MDA content was increased,which was 1.70 and 1.48 times higher than that in the control group and 1.79 and 1.55 times higher than that in the BBWE group,respectively,and the differences were statistically different（P<0.05）.Meanwhile,compared with the SOD content of the control group（175.01 U/mg protein）,the cellular SOD content of the 0.8mg/m L BBWE group,4μmol/L CdSO₄group and 0.8 mg/m L BBWE+4μmol/L CdSO₄group were 210.82 U/mg protein,122.73 U/mg protein and 131.43 U/mg protein,and the SOD content of cells in both the CdSO₄group and the co-induced group was reduced（P<0.05）.Conclusions1、With the increase of CdSO₄exposure concentration to NIT-1 cells of pancreaticβcells,the intracellular antioxidant enzymes gradually decreased,and the lipid peroxide MDA increased.Under the stimulation of high glucose,the cell insulin secretion decreased,and the intracellular ROS level continued to rise,which eventually led to apoptosis.2、In damaged NIT-1 cells,the level of m⁶A modification may be jointly regulated by METTL3 and FTO,and the level of m⁶A modification is positively correlated with m⁶A-modified LncRNAs（LncRNA-MALAT1,LncRNA-PVT1）in injured cells.3、Bombyx Batryticatus Water-soluble Extracts at a low concentration can promote the proliferation of NIT-1 cells,but it cannot antagonize the cell damage induced by CdSO₄.Instead,it enhances the cytotoxicity,which is manifested by increased intracellular ROSlevels,increased MDA content,and decreased SOD content The mechanism may be related to the depletion of antioxidant enzymes in the cell,apoptosis and the increase of ROS levels.