The Effect And Mechanism Of Tagetes Erecta L.Extract On Arsenic-induced Pancreatic β Cell Function Damage

Objective（1）To observe the effects of arsenic trioxide（As₂O₃）on the survival rate,apoptosis and oxidative damage of mouse insulinoma beta cells（NIT-1）;（2）To understand the effect of marigold extract on NIT-1 cells,to determine whether marigold extract can affect the damage effect of As₂O₃on NIT-1 cells,and to preliminarily explore the mechanism from the perspective of oxidative stress;（3）The changes of nuclear transcription factor NF-E2-related factor（Nrf2）and long non-coding RNA in the function injury of NIT-1 cells induced by As₂O₃induced by marigold extract were analyzed.MethodNIT-1 cells were taken as the subject.Firstly,the cells were treated with As₂O₃and marigold extract at different concentrations,and then the cells were treated with different combinations of As₂O₃and marigold extract to carry out the following experiments:（1）Construction of pancreatic isletβ-cell injury model:CCK-8 method was used to detect the cell survival rate of As₂O₃and marigold extract treated alone or combined with the two treatments.Hoechst staining method could observe the cell morphology and degree of apoptosis after the combined treatment with the two treatments by microscope,and calculate the cell apoptosis rate;（2）the accumulation of reactive oxygen species and oxidative stress level detection:fluorescent probe method with fluorescence microscopy and quantitative detection of the NIT-1 cells after combined treatment of reactive oxygen species（ROS）level,the WST-8 of NIT-1 method for testing the super oxide dismutase（SOD）activity,colorimetric method of malondialdehyde（MDA）content for testingy;（3）Detection of changes in Nrf2 signaling pathway:NIT-1 cells mRNA levels of Nrf2 and HO-1,lnc RNA036805 and lnc RNA029382 were detected by polymerase chain reaction（RT-PCR）after combined treatment Results（1）Cell survival rate and apoptosis:Observed under the microscope,cells without the effect of As₂O₃are intact.As the concentration of As₂O₃increases,NIT-1 cells gradually lose their ability to adhere to the wall and float in the culture medium.CCK-8experiment results showed that the survival rate of NIT-1 cells after being infected with0,0.4,0.8,1.6,3.2,6.4 and 12.8 mg/L As₂O₃for 24 hours was 100%,96.11%,93.79%,74.92%,63.02%,52.75%,42.22%,the cell survival rate showed a trend of gradualdecline with the increase of As₂O₃concentration（F=258.545,P<0.001）;the cell morphology changes under the treatment of 0.04 and 0.08 mg/m L marigold extract Obviously,0.16,0.32,0.64,1.28 mg/m L marigold extracts gradually lose their ability to adhere to the wall and float in the culture medium.Further CCK-8 test results show that NIT-1 cells have undergone 0,0.04,After the concentration of 0.08,0.16,0.32,0.64,1.28 mg/m L marigold water extract was used for 24 hours,the cell survival rate was100%,104.05%,109.43%,92.47%,77.36%,49.44%,22.08%,and the survival rate showed The trend of rising first and then falling（F=190.206,P<0.001）;after0.08mg/m L marigold and 3.2mg/LAs₂O₃,the cell survival rate was 34.64%,which was lower than the“0”group and the marigold group（105.93%）,As₂O₃group（59.40%）;3.2mg/L As₂O₃treated cells for 6h and then 0.08mg/m L marigold for 24h,the detected cell survival rate was 66.53%,which was significantly higher than As₂O₃group（56.54%）;then The apoptotic rate of each group designed for post-treatment of marigold was determined.The“0”group,the marigold group,the As₂O₃group and the3.2mg/L As₂O₃+0.08mg/m L marigold water extract post-treatment group（hereinafter referred to as the combined Treatment group）The apoptosis rate was 8.67%,5.5%,21.33%,and 17.17%.The apoptosis rate of the combined treatment group was lower than that of the As₂O₃group and the single staining group.The difference was statistically significant（P=0.027）.（2）Nrf2 signaling pathway related indicators and oxidative stress related indicators:After NIT-1 cells were treated with marigold,As₂O₃and the combined treatment group for 24 hours,the m RNA level of HO-1 in the As₂O₃single staining group and the combined treatment group was significantly higher than The“0”group was 21.65 and8.17 times that of the“0”group(P_As2O3<0.001,P_combined=0.043).The m RNA level of HO-1 in the marigold group was 0.95 times that of the control group,and the difference was not significant（P=0.986）);The m RNA level of HO-1 in the combined treatment group was significantly higher than the“0”group and marigold group,but lower than the As₂O₃group,the difference was statistically significant(P₀=0.043,P_wan=0.02,P_As2O3=0.041).Comparison of Nrf2 m RNA levels among the“0”group,marigold group,and combined treatment group,the difference was statistically significant（F=19.179,P=0.001）,the Nrf2 m RNA expression level of the As₂O₃group was 3.05 of that of the“0”group The difference was statistically significant（P<0.001）;the m RNA levels of the marigold group and the combined treatment group were 0.90 and 1.42 times that of the“0”group,and the difference was not statistically significant(P_wan=0.769,P_combined=0.231);Comparing the combined treatment group（1.42）with the As₂O₃group（3.05）,the m RNA level of Nrf2 was significantly lower than that of the As₂O₃group（P=0.001）.Compared with the combined treatment group（1.42）and the marigold group（0.90）,the difference was not significant(P₀=0.231,P_wan=0.148);the detection results of intracellular ROS content showed that the ROS activity comparison between the groups was statistically significant（F=66.026,P<0.001）,and the ROS activity in the marigold group was that of the“0”group 0.97 times（P=0.035）;the ROS activity of the As₂O₃group and the combined treatment group was 1.12 and 1.06(P_As2O3<0.001,P_combined=0.001)of the“0”group,and the ROS activity of the combined treatment group was lower than that of the As₂O₃group,but higher than that of the“0”group.The difference between the“0”group and the marigold group was statistically significant(P₀=0.001,P_wan<0.001,P_As2O3=0.001);the SOD activity of the“0”group,marigold group,As₂O₃group and combined treatment group was 85.18±0.88,95.99±24.99,69.66±3.90 and 76.82±5.30 U/mg Protein,the difference in SOD activity between the groups was statistically significant（F=27.963,P<0.001）,and the SOD activity of the combined treatment group was significantly higher than The As₂O₃group was lower than the marigold group(P_As2O3=0.046,P_wan<0.001);the MDA content of the“0”group,the marigold group,the As₂O₃group and the combined treatment group were2.71±0.042,2.11±0.079,3.78±0.048 and 3.13±0.035 nmol/mg protein,the difference in MDA content between the groups was statistically significant（F=29.096,P<0.001）.The MDA content of the combined treatment group was higher than that of the marigold group(P_wan=0.001),but it was low In the As₂O₃group(P_As2O3=0.008);（3）Lnc RNA levels:The measurement results of th P_As2O3e levels of Lnc RNA036805 and Lnc RNA029382 showed that the Lnc RNA036805 levels of the marigold group,the As₂O₃group,and the combined treatment group were 4.69,0.104and 0.205 times that of the“0”group,respectively.The difference was statistically significant（F=130.425,P<0.001）Compared with the other three groups,the level of Lnc RNA036805 in the combined treatment group was significantly lower than that of the“0”group and the marigold group(P₀=0.018;P_wan<0.001),but higher than that of the As₂O₃group,the difference was not significant(P_As2O3=0.717);Lnc RNA0293822level,marigold group,As₂O₃group,combined treatment group were 3.60,42.49 and8.76 times of the combined treatment group of“0”group,the difference was significant（F=30.614,P<0.001）.Compared with the other three groups,the level of Lnc RNA0293822 was lower than that of the As₂O₃group,the difference wasstatistically significant(P_As2O3<0.001),but it was higher than the“0”group and the marigold group,the difference was not statistically significant(P₀=0.154;P_wan=0.326).Conclusion（1）Marigold extract stimulates cell growth at low concentrations,while high concentrations reduce cell survival.As₂O₃can reduce cell survival and induce apoptosis in a concentration-dependent manner.Marigold post-treatment can effectively antagonize As₂O₃.Decrease cell survival rate and cell apoptosis;（2）As₂O₃exposure can increase the m RNA levels of Nrf2 and HO-1,and the levels of oxidative stress indicators such as SOD,MDA,and ROS increase.Post-processing of marigold extract can effectively reverse this increase,suggesting that marigold can be used to a certain extent Alleviate the oxidative stress caused by arsenic exposure;（3）As₂O₃exposure caused the level of Lnc RNA029382 to rise,and the level of Lnc RNA036805 to decrease.The post-processing of marigold extract can effectively antagonize the changes of Lnc RNA029382 and Lnc RNA036805 levels caused by As₂O₃exposure.