| ObjectivesMulti-walled carbon nanotubes(MWCNTs)are currently the most widely used new types of nano-fiber materials.Whether they can have identical harmful effects similar to that caused by asbestos has been highly concerned because of their similarity with asbestos in fiber characteristics and exposure methods.Existing studies have shown that MWCNTs can induce mesothelioma in animals(it was classified as a category 2B carcinogen by the International Agency for Research on Cancer in 2017),which poses a great potential threat to human health,but its mechanism of action is unknown.China is one of the largest producers and consumers of MWCNTs.Therefore,studying the molecular mechanism of pleural mesothelioma caused by MWCNTs can provide a scientific basis for more accurate evaluation of its safety and the development of effective prevention and treatment measures,which undoubtedly has important scientific and practical significance.The inflammatory inducing effect of MWCNTs is one of the most recognized major toxic effect mechanisms at present.Chronic inflammation is an important factor in the development of pleural mesothelioma caused by asbestos.The chronic inflammation in the pleural cavity is also a significant contributor to MWCNTs-induced pleural mesothelioma in mice.Therefore,how chronic inflammation play roles and which ways are involved in the process of pleural mesothelioma caused by MWCNTs,this is the key of the study about the molecular mechanism of MWCNTs-induced pleural mesothelioma.However,the following problems exist in the study of the mechanism of chronic inflammation in MWCNTs-induced pleural mesothelioma: Firstly,the animal experiments were injected with high-dose,one-time of MWCNTs.MWCNTs-induced pleural mesothelioma is a long-term,low-dose sustained process.One-shot injection cannot simulate the actual exposure of MWCNTs in the ways of exposure route and exposure dose.Secondly,experiments in vitro mostly use single-cell culture and cannot simulate the cellular environment in the pleural cavity.Only if the experimental system based on the actual exposure dose and exposure environment could elucidate the molecular mechanism of MWCNTs-induced mesothelioma.Therefore,our study explore the role of chronic inflammatory response in malignant transformation of pleural mesothelial cells induced by MWCNTs and its molecular mechanism by the way of low-dose and long-term exposure and co-culture system of human pleural mesothelial cells and macrophage cells,hoping to provide a scientific basis for occupational safety protection of nanomaterials practitioners,and provide new ideas and methods for the treatment of mesothelioma.MethodsThe experiment included four groups: mesothelial cells,macrophage + mesothelial cells,MWCNTs + mesothelial cells,and MWCNTs + macrophage + mesothelial cells;the final concentration of MWCNTs we used was 0.1μg/mL;the period was 3 months;and the following experiments were used for our study:1.Scanning electron microscopy(TEM)and transmission electron microscopy(TEM)were used to investigate whether the MWCNTs used conformed to the precondition of “pathological fiber”;2.By comparing the expression of cytokines among different groups,we investigated whether MWCNTs could induce inflammatory reaction and the interaction between mesothelial cells and macrophages.3.The role of inflammatory cells in the malignant transformation of human pleural mesothelial cells induced by MWCNTs was investigated by cell proliferation,cell migration,cell invasion,colony formation assay,chromosomal aberration assay,and animal tumor formation assay.4.The transcriptome sequencing technology was used to screen the candidate genes involved in the malignant transformation of human pleural mesothelial cells induced by MWCNTs.The candidate genes were verified by qRT-PCR and western blot techniques,and the possible molecular mechanism was proposed.5.Construct a pleural mesothelial cell line with stable knockdown of candidate genes and co-culture it with macrophages in the presence of MWCNTs,and compare the ability of cell proliferation,migration,invasion,and colony formation between different groups to examine the roles of candidate genes in malignant transformation of mesothelial cells caused by MWCNTs;6.Construct a pleural mesothelial cell line with stable knockdown of candidate genes and determine the protein expression level and the interaction between them in the mesothelial cells(with stable knockdown of candidate genes)+ MWCNTs + macrophage group by western blot.ResultsThrough the above experiments,we have obtained the following results:1.The average length of MWCNTs used in this experiment is 10-20μm and the average tube diameter is 30-50 nm,which is consistent with the characteristics of pathological fibers.2.MWCNTs can cause significantly high expression of IL-1β cytokines by macrophages;there is close relationship between macrophages and mesothelial cells: IL-1β cytokines secreted by macrophage can enhance the release of inflammatory factors such as IL-8,TNF-α and IL-6 in mesothelial cells induced by MWCNTs.3.Long-term and low-dose of MWCNTs can induce the chromosomal aberration of mesothelial cells in vitro;In the co-culture system of MWCNTs+macrophages+ mesothelial cells,the chromosomal aberration rate of mesothelial cells was higher than those of other groups,and the difference was statistically significant.This result shows that MWCNTs can increase the chromosomal aberrations rate of mesothelial cells and macrophages may promote it.4.Long-term and low-dose of MWCNTs can induce the malignant transformation of mesothelial cells in vitro;In the co-culture system of MWCNTs+macrophages+ mesothelial cells,the proliferation,migration,invasion and colony formation ability of mesothelial cells were significantly higher than those of other groups,and the difference was statistically significant.This result indicates that macrophages have significant role in the malignant transformation of mesothelial cells caused by MWCNTs.5.Nude mice with subcutaneous inoculation with mesothelial cells in the co-culture system of MWCNTs+ macrophages+mesothelial cells showed a significantly higher tumor volume and Ki67-positive rate in the tumor-bearing tissues than other groups,and the difference was statistically significant.The results suggest that the malignant transformation of mesothelial cells co-cultured with macrophages is higher.6.Through transcriptome sequencing,a series of differentially expressed genes were screened.Combined with the previous results,NF-κB,IL-6,and STAT3 were considered to be most closely related to the malignant transformation of mesothelial cells induced by MWCNTs;Results of qRT-PCR and western blot showed that the expression of NF-κB(p65),IL-6,STAT3 in mRNA and protein in MWCNTs+macrophages+mesothelial cells group was significantly higher than those in other groups.These results suggest that NF-κB(p65),IL-6 and STAT3 may play a role in the malignant transformation of pleural mesothelial cells induced by MWCNTs.7.The proliferation,migration,invasion and colony formation of mesothelial cells in the group with mesothelial cells(sh-p65 or sh-IL-6R or sh-STAT3)+MWCNTs+ macrophage were significantly lower than that of mesothelial cells(sh-NC)+MW CNTs+macrophage group,and the difference was statistically significant.The results demonstrate that NF-κB,IL-6 and STAT3 play an important role in the malignant transformation of pleural mesothelial cells induced by MWCNTs.8.The expression of IL-6,STAT3 protein was decreased after knock-down of NF-κB(p65),and the expression of NF-κB(p65)protein was unchanged and the expression of STAT3 protein was decreased after knock-down of IL-6R,and the expression of NF-κB(p65)and IL6 R protein remained unchanged after knock-down of STAT3 in co-culture system of mesothelial cells(sh-NC or sh-p65 or sh-IL-6R or sh-STAT3)+MWCNTs+ macrophages.This result indicates that there exists NF-κB/IL-6/STAT3 molecular cascade in the malignant transformation of mesothelial cells induced by MWCNTs,and the NF-κB/IL-6/STAT3 pathway plays an important role.ConclusionsBased on the above studies,we made conclusions as follows:1.IL-1β secreted by macrophages stimulated by MWCNTs can significantly enhance the release of inflammatory cytokines in mesothelial cells.2.MWCNTs can induce the malignant transformation of mesothelial cells at long-term and low-dose exposure.3.Inflammation can enhance the malignant transformation of pleural mesothelial cells induced by MWCNTs.4.NF-κB/IL-6/STAT3 pathway plays an important role in the process that inflam matory response promotes the malignant transformation of pleural mesothelial cells induced by MWCNTs. |
PDF Full Text Request