Glyburide Attenuates B(a)p And LPS-induced Inflammation-related Lung Tumorigenesis In Mice By Inhibiting NLRP3 Inflammasome

ObjectiveGlyburide(Gly)is one of the most common medication used in the treatment of type 2 diabetes,and it serve as inhibitor of the NLRP3 inflammasome.Accumulating evidence suggests that Gly can decrease cancer risk and tumor growth,and also can inhibite proliferation and migration of lung cancer cell.However,the role of chronic Gly treatment in inflammation-related lung cancer induced by Benzo(a)pyrene[B(a)p]plus lipopolysaccharide[LPS]and its mechanisms remain unclear.In this study,we established the model of inflammation-related lung cancer in B(a)P and LPS induced mice with and without Gly,evaluated whether Gly inhibite the occurrence of lung cancer and explored the possible mechanisms,for providing preventive and treatment strategies for inflammation-related lung cancer.Methods1.The specific pathogen free(SPF)wide type of C57BL/6J mice were divided into six groups randomly:vehicle control group(n=16),B(a)p plus LPS group(n=30),Gly(0.48mg/kg)-treated tricaprylin group(Tricaprylin+Gly(0.48mg/kg),n=19),Gly(0.48mg/kg)-treated B(a)p plus LPS group(B(a)P/LPS+Gly(0.48mg/kg),n=31),Gly(0.96mg/kg)-treated tricaprylin group(Tricaprylin+Gly(0.96mg/kg),n=16),Gly(0.96mg/kg)-treated B(a)p plus LPS group(B(a)P/LPS+Gly(0.96mg/kg),n=34)The mice in B(a)p plus LPS group were exposed to B(a)p(lmg/mouse,dissolved in 50?l tricaprylin,once a week for 4 times,the week of the last time of B(a)p treatment named Week 0)and 3 weeks later instilled intratracheally with LPS(2.5?g/mouse,dissolved in 50?l saline)once every three weeks for 5 times,tricaprylin and saline were used for vehicle controls.The Gly was administered by gavage(at a dose of 10?l/g body weight)one week before the mice exposed to B(a)p three times a week(Monday,Wednesday,and Friday)until the animal model finished in Gly-treated tricaprylin group and Gly-treated B(a)p plus LPS group.2.Fasting blood glucose(FBG)of Gly-treated mice were detected about once a month in the whole animal model established.And comparison average of weight change in mice of all groups on 190th day.At week 22,a batch of mice were selected as the first stage of animal model,the rest of mice regarded as second stage of the animal model.All the mice were sacrificed,the lung tissues were removed and weighed to estimate lung coefficient and the incidence and the mean tumor count of lung tumors were analyzed.The left lobes of the lungs of the second stage mice were fixed in 4%paraformaldehyde for observation histopathological change of lung tissues by HE.3.The expression of NLRP3 protein,IL-1? and IL-18 protein in lung tissue was detected by immunohistochemical SP method.Western blotting was used to detecte the expression of Cleaved-IL-1?.Results1.Mice were fed normally for 22 weeks,Vehicles treatment and Gly plus tricaprylin administration could not induce lung tumorigenesis.However,tumors were observed in B(a)p plus LPS group,B(a)P/LPS+Gly(0.48mg/kg)group and B(a)P/LPS+Gly(0.96mg/kg)group,the incidence of lung tumor were 40%,37.5%and 70%respectively,and the mean tumor count of these groups were(2.5±1.77),(1.88±1.34)and(3.3±0.87),but there were no significant difference of the incidence of lung tumor and mean tumor count among these groups.At week 30,lung tissues of the mice were removed and observed.No visible tumors were observed in vehicle control group,Tricaprylin+Gly(0.48mg/kg)group and Tricaprylin+Gly(0.96mg/kg)group.The incidence of lung tumor in B(a)P plus LPS group was 89.4%,treated with 0.48mg/kg or 0.96mg/kg Gly,the incidence of lung tumor decreased to 82.6%and 70.8%respectively.The mean tumor count of mice in B(a)P/LPS+Gly(0.96mg/kg)group(2.13±0.42)was significantly decreased compared to those in B(a)p plus LPS group(5.581±1.04)(P<0.05),whereas there was no significant difference between B(a)P/LPS+Gly(0.48mg/kg)group(3.22±0.63)and B(a)p plus LPS group.2.The lung coefficient of mice was no significant difference in the first stage.In the second stage,the lung coefficient of mice in B(a)p plus LPS group significantly increased(P<0.05)compared with Vehicle control group,administered with 0.96mg/kg Gly the lung coefficient of mice in B(a)p plus LPS group significantly decreased(P<0.05).The mice treated with 0.48mg/kg or 0.96mg/kg Gly,FBG from the 112th day or 62th day until the end of animal model decreased significantly compared with the FBG of 1th day,respectively.Average weight of the mice treated with 0.48mg/kg or 0.96mg/kg Gly increased slowly,compared with vehicle control group,the average of weight change of mice in Tricaprylin+Gly(0.48mg/kg)group and Tricaprylin+Gly(0.96mg/kg)group reduced significantly(P<0.05),and Tricaprylin+Gly(0.9 6mg/kg)group decreased significantly than Tricaprylin+Gly(0.48mg/kg)group(P<0.05);Average of weight change of mice in B(a)P+LPS group,B(a)P/LPS+Gly(0.48mg/kg)group and B(a)P/LPS+Gly(0.96mg/kg)group lower than vehicle control group(P<0.05),B(a)P+LPS group and B(a)P/LPS+Gly(0.96mg/kg)group significantly decreased compared with B(a)P/LPS+Gly(0.48mg/kg)group(P<0.05).3.The mice in the second stage,histopathological examination revealed a reduction of the number of pathological tumor nest in the B(a)P/LPS+Gly(0.48mg/kg)group(0.38±0.18)and B(a)P/LPS+Gly(0.96mg/kg)group(1.31±0.35)compared to the B(a)p plus LPS group(3.56±1.02)(P<0.05),the lung inflammation was improved after Gly treatment.4.The expression of NLRP3 and IL-1? protein was significantly increased in B(a)p plus LPS group compared to vehicle control group(P<0.05,respectively),and the expression of NLRP3 and IL-1? protein was significantly decreased in B(a)P/LPS+Gly(0.96mg/kg)group compared to B(a)p plus LPS group(P<0.05,respectively).However,there was no significantly difference of the expression of IL-18 protein in B(a)p plus LPS groups,B(a)P/LPS+Gly(0.48mg/kg)group and B(a)P/LPS+Gly(0.96mg/kg)group.In addition,the expression of Cleaved-IL-1? also significantly increased in B(a)p plus LPS group compared to vehicle control group(P<0.05),treated with 0.96mg/kg Gly the expression of Cleaved-IL-1? significantly decreased(P<0.05).ConclusionsGlyburide attenuates B(a)P and LPS-induced inflammation-related lung tumorigenesis in mice by inhibiting NLRP3 inflammasome.