Study On Maneb-induced Nerve Cellular Senescence And The Protective Effects And Mechanisms Of Procyanidins

During the process of human aging,the brain is one of the first organs to age.In the process of physiological aging,a series of changes take place in the structure and function of the brain.Under the combined action of some internal and external factors,the brain gradually changes from physiological senescence to pathological senescence,which eventually leads to the occurrence of neurodegenerative diseases.At present,the pathogenesis of neurodegenerative diseases is still not very clear,and clinical treatment is limited.At present,it is generally believed that aging is the biggest risk factor for neurodegenerative diseases.Therefore,protective strategies for aging may be an effective plan to inhibit the occurrence and development of neurodegenerative diseases.Maneb,the agricultural fungicide,is widely used in agricultural production and harmed to the environment and human beings.This study firstly explored the Manebinduced cellular senescence and the related molecular mechanism in human neuroblastoma cell SH-SY5Y.MTT assay was used to detect the effect of Maneb on the cell activity of SH-SY5Y cells at three time gradients of 24h,48h and 72h.The results showed that Maneb had a toxic effect on SH-SY5Y cells and inhibited the cell activity in a time and dose-dependent manner.The S A-β-Gal staining experiment found that under the same concentration of Maneb treatment,the number of blue cells gradually increased with the time course,indicating that the activity of β-galactosidase related to senescence increased,that is,the numbers of senescent cells increased.According to the characteristics of senescent cells,we further detected the content of ROS.SH-SY5Y was treated with Maneb(0-200 μM)for 24 hours,and then treated on the basis of the kit.The results of flow cytometry showed that,Maneb reduced the ROS level of SH-SY5Y in a dose-dependent manner.Further,we analyzed the cell cycle distribution and found that SH-SY5Y cells were arrested in S phase after treatment with Maneb in a dose-dependent manner.After JC-1 staining,the results under the microscope showed that the MMP gradually decreased in a dose-dependent manner.The results of flow cytometry were consistent with those observed under the microscope.In addition,we also conducted experiments on the molecular mechanism.Western blot experiments showed that with the increase of Maneb concentration,the expressions of senescence-associated proteins p16 and Rb increased in a dosedependent manner.We also detected the upstream proteins p38 and ERK of p16,and found that the expression levels of p38 and ERK also increased with the increase of contemporary Maneb concentration.Then,we conducted RT-PCR experiments from the RNA level,and PCR results showed that after Maneb treatment,the mRNA expression levels of p16 and Rb were gradually increased in a dose-dependent manner.Comprehensive experiments showed that Maneb,an agricultural fungicide,induced senescence in human neuroblastoma cells SH-SY5Y through the p16-Rb signaling pathway.Our experiments are just the tip of the iceberg,and there are many more aspects that need to be further studied.The most important thing is to develop drugs that inhibit senescence and reduce the suffering caused by neurodegenerative diseases.Next,the protective effect of procyanidine on the senescence of SH-SY5Y cells induced by Maneb was explored.We first tested the cytotoxicity of procyanidine by MTT method.The experiment showed that procyanidine were less toxic to human neuroblastoma SH-SY5Y cells,and almost non-toxic.Then we continued to detect the protective effect of procyanidine by MTT method.The experiment showed that procyanidine could reduce the toxicity of Maneb to SH-SY5Y cells.We first pretreated SH-SY5Y cells with different concentrations(0-50 μM)of procyanidine for 30 min,and then treated them with Maneb for 24 h,and then treated them according to the instructions of the aging detection kit.From the observation,it can be seen that with the increase of procyanidine concentration,the number of blue cells gradually decreased,that is,the activity of SAβ-Gal gradually decreased,and the numbers of senescent cells gradually decreased.The SH-SY5Y cells were treated in the same way,followed by ROS kit for post-processing,and flow cytometry was used to detect each group.The experiment showed that with the increase of procyanidine concentration,ROS content decreased.After the same preliminary treatment,the distribution of cell cycle was detected,and the results of flow cytometry showed that the proportion of S phase gradually decreased with the increase of OPC concentration.The results of JC-1 staining showed that the mitochondrial membrane potential of SH-SY5Y cells increased gradually with the increase of OPC concentration,which was consistent with flow cytometry results,indicating that OPC increased the mitochondrial membrane potential of SH-SY5Y cells treated with Maneb.Consistently,the above results shown that procyanidine increased the mitochondrial membrane potential of SH-SY5Y cells after Maneb treatment.In addition,western blotting showed that with the increase of OPC concentration,the expression levels of p16,Rb,p38 and ERK decreased.We preliminarily demonstrated that procyanidine have a protective effect on the senescence effect of Maneb-induced human neuroblastoma SH-SY5Y cells.This study may provide some ideas for the treatment of neurodegenerative diseases,but also has certain shortcomings and limitations,and further research is needed.