The Study On The Role Of NLRP3 In The Process Of Resveratrol Inhibiting Renal Cell Carcinoma

ObjectiveResveratrol(RSV)is a natural polyphenolic compound containing in a variety of plants,such as grapes,berries and peanuts.RSV has been reported to exert anti-inflammatory,antioxidant and anti-tumor function,and it also can resist obesity,protect cardiovascular system and alleviate Alzheimer’s disease(AD).Renal Cell Carcinoma(RCC)is one of the most common urologic malignant tumor.Because the pathogenesis of RCC is not fully understood and it is insensitive to chemotherapy,the patients’ prognosis is poor.It is known that inflammation is closely related to the tumorigenesis and progress,and it has the mutual promotion effect.Inflammasomes,which play an important role in the process of inflammation,have been extensively studied.Among them,the NLRP3 inflammasome is the most well known,however,its role in the development of RCC remains unclear.We hope to provide new ideas for clinical therapy through observing the effect of RSV on RCC and evaluating NLRP3 expression level in the process.Therefore,the purpose of this project is not only to study the effect of RSV on RCC and to observe the changes of inflammation in this process to reveal the role of NLRP3 in the development of RCC,but also to provide new clues for studying the relationship between inflammation and tumor.Methods 1.CCK8 assay measured the effect of RSV on the viability of ACHN and 786-O cells.2.Wound healing assay measured the effect of RSV on the migration ability of ACHN and 786-O cells.3.Transwell assay measured the effect of RSV on the invasion ability of ACHN and 786-O cells.4.Flow cytometry analysis measured the effect of RSV on the apoptosis of ACHN and 786-O cells.5.Tumor xenograft model was established and randomly divided into two groups:CON group received daily resveratrol administration 25% alcohol(60 mg/kg,gavage injection)and RSV group which were received daily resveratrol administration(60 mg/kg,gavage injection),and the condition of nude mice was observed every day,including survival and body weight of nude mice.6.The tumor volumes were measured by a caliper regularly,and the tumor weight were measured by scales after removing the tumor on day 40.These data were recorded and analyzed.7.TUNEL staining and Western blot analysis measured the apoptosis in tumor issues.8.Immunohistochemical staining and Western blot analysis measured the expression level of NLRP3 in tumor tissues.9.Western blot analysis measured the nlrp3 silencing efficiency.10.Wound healing assay measured the effect of RSV and NLRP3 on the migration ability of ACHN cells.11.Transwell assay measured the effect of RSV and NLRP3 on the invasion ability of ACHN cells.12.Western blot analysis measured protein levels of Sirt1,NLRP3 and Bax in ACHN cells after treatment with RSV and NLRP3 siRNA.Real-Time PCR measured mRNA levels of NLRP3.Western blot analysis measured protein levels of Sirt1 in tumor tissues.Results 1.CCK8 assay showed RSV inhibited growth capacity of ACHN and 786-O cells in a time-and dose-dependent manner.2.Wound healing assay showed RSV suppressed the migration ability of ACHN and 786-O cells.3.Transwell assay showed RSV suppressed the invasion ability of ACHN and 786-O cells.4.Flow cytometry analysis showed RSV promoted the apoptosis of ACHN and 786-O cells.5.Results showed that RSV improved the ratio of survival in tumor-bearing mice but didn’t affect the mice body weight.6.Data showed RSV inhibited tumor growth.7.TUNEL staining and Western blot analysis showed RSV promoted tumor apoptosis.8.Immunohistochemical staining and Western blot analysis showed RSV decreased the expression level of NLRP3 in tumor tissues.9.Western blot analysis showed the nlrp3 silencing efficiency was around 66.17% in ACHN cells.10.Wound healing assay showed silencing NLRP3 suppressed the migration ability of ACHN cells.11.Transwell assay showed silencing NLRP3 suppressed the invasion ability of ACHN cells.12.Western blot analysis showed Sirt1 was activated in vivo not in vitro,and RSV increased the level of Bax to facilitate apoptosis.Western blot analysis and Real-Time PCR showed that the expression level of NLRP3 was declined when treated with RSV and NLRP3 siRNA alone or combination compared with CON group.Conclusions 1.RSV can inhibit renal carcinoma cells growth and promote them apoptosis.2.RSV can inhibit migration and invasion capacities of renal carcinoma cells,and the process was down-regulated by NLRP3.3.Furthermore experiments in vivo demonstrated that RSV can inhibit renal carcinoma growth and promote them apoptosis,and can decrease the expression of NLRP3 inflammasome,leading to improving the ratio of survival in tumor-bearing mice.