【作者】 王超；

【导师】 于录；

【作者基本信息】 吉林大学 ， 兽医公共卫生， 2020， 博士

【摘要】 随着世界经济迅速增长,人类的生活习惯和所处的生存环境发生了巨大变化,随之而来的各类疾病给人类造成了巨大伤害,特别值得注意的是,地球是动物和人类共同的家园,环境的变化给人类造成疾病的同时动物也难以幸免,其中,由于癌症的发生特质被认为是对人和动物危害性最大的全球性疾病。在2018年全球大约有1808万癌症的新发病例和956万癌症死亡病例,同时,每年约有20%-30%的宠物犬和猫是死于各种原因导致的癌症,造成的经济损失高达上百亿元。据统计,动物和人类的癌症发病种类高达上百种,且都具有类似的共同特征,其中癌症引发的炎症反应是癌症恶病质状态和复发的主要原因,癌症相关的炎症可以促进新血管的生成和癌细胞转移。炎症反应是机体为消灭来自于体内外的异物而产生的一系列复杂的生物学反应。活性氧(ROS)作为在炎症反应和癌症的发生发展中都占有重要地位的一种细胞产物,自然地将炎症反应和癌症联系在一起。ROS是机体细胞在正常代谢过程中产生的一种副产物,而当细胞受到外界刺激时ROS的产量会急剧升高,从而导致一系列的生物学反应。NF-κB是一种氧化应激传感器调控多种细胞功能,包括炎症信号传导,且NF-κB的异常激活与癌症的发生发展相关。NLRP3炎症小体是一种重要的炎症反应调节因子,被激活后会产生炎症级联反应,并且能够促进癌症的发生、发展和转移。胰岛素样生长因子1(IGF-1)是一种哺乳动物的生长因子类似物,在婴儿的生长和成人体内持续进行合成代谢,有报道表明IGF-1能够调节炎性因子的表达,并且IGF-1与多种癌症的发生发展有关,但是其作用机制尚未见报道。针对IGF-1在炎症反应和癌症中的重要作用,在本研究中,我们以人宫颈癌细胞(HeLa)、犬骨肉瘤细胞(D-17)和小鼠黑色素瘤细胞(B16-F10)为模式细胞,与人、犬和小鼠正常细胞(NCM-460细胞、MDCK细胞和MODE-K细胞)在体外进行对比研究,并结合动物体内荷瘤实验,探究IGF-1是否通过激活经典的NF-κB和NLRP3通路介导癌细胞的炎症反应发生,最关键的是,我们将在国内外率先探究IGF-1调节NF-κB和NLRP3炎症通路激活的上游分子机制是什么,从而发现IGF-1调节癌细胞发生炎症的通路关键蛋白并找到其抑制剂,最后给出抑制癌症相关炎症发生的解决方案,以期实现癌症病人和动物带瘤生存。首先,我们考察IGF-1能否诱导人、犬和小鼠三种癌细胞(HeLa细胞、D-17细胞和B16-F10细胞)发生炎症反应表型。利用荧光酶标仪对三种癌细胞ROS的产生情况进行检测;使用qPCR技术和ELISA方法对IGF-1诱导三种癌细胞细胞以及正常细胞(NCM-460细胞、MDCK细胞和MODE-K细胞)炎性因子IL-1β、IL-6和TNF-α的分泌情况进行检测。结果表明:IGF-1能够诱导三种癌细胞的细胞质中ROS产生,且最适诱导浓度均为100 ng/mL,最适诱导时间均为120分钟;线粒体ROS抑制剂(MitoQ)对IGF-1诱导三种癌细胞的ROS产量没有影响,而NOX2抑制剂(DPI)能显著抑制IGF-1诱导三种癌细胞的ROS产量,这表明IGF-1诱导三种癌细胞产生的ROS主要来自于细胞质并受NOX2介导;IGF-1能够诱导三种癌细胞分泌的炎性因子IL-1β、IL-6和TNF-α增多及其mRNA的表达;而ROS清除剂(NAC)能显著抑制IGF-1诱导三种癌细胞中IL-1β、IL-6和TNF-α的mRNA表达以及细胞上清中三种炎性因子的分泌。并且我们发现相同处理条件下,IGF-1不能诱导人、犬和小鼠正常三种细胞ROS产量升高和上述炎症细胞因子的分泌量升高。综上结果表明,IGF-1能够诱导人和动物癌细胞出现炎症反应表型,但不能诱导正常细胞出现炎症反应表型,所以值得进一步深入研究IGF-1介导癌细胞炎症反应发生的机制。其次,针对人、犬和小鼠癌细胞,并以正常细胞作对照,我们探索了IGF-1是否通过激活经典的NF-κB和NLRP3通路介导癌细胞的炎症反应。借助Western blot检测IGF-1处理后人、犬和小鼠的癌细胞(HeLa细胞、D-17细胞和B16-F10细胞)以及正常细胞(NCM-460细胞、MDCK细胞和MODE-K细胞)中NF-κB和NLRP3相关炎症通路蛋白的表达情况;通过qPCR和ELISA方法对在NF-κB抑制剂BAY 11-7082和Caspase 1抑制剂Ac-YVAD-cmk作用下各种癌细胞中IL-1β、IL-6和TNF-α的表达量进行检测。结果表明,IGF-1能够诱导人、犬和小鼠三种癌细胞中炎症通路蛋白IKK、NF-κB、NLRP3、IL-1β、ASC和Caspase 1的高表达,并且ROS清除剂NAC能够显著抑制IKK、NF-κB、NLRP3、IL-1β、ASC和Caspase 1的表达;NF-κB抑制剂和Caspase 1抑制剂能够显著抑制IGF-1诱导三种癌细胞中IL-1β、IL-6和TNF-α的mRNA表达以及细胞上清中三种炎性因子的分泌;与三种癌细胞相同处理条件下,IGF-1不能诱导正常细胞中的NF-κB和NLRP3相关炎症通路蛋白表达的变化。综上结果表明:IGF-1能够诱导人、犬和小鼠的癌细胞中的NF-κB和NLRP3中相关炎症通路蛋白的表达,不能激活正常细胞的相应通路蛋白的表达。综上所述,IGF-1能够通过激活经典的NF-κB和NLRP3通路介导癌细胞的炎症反应发生。再次,针对癌症模式细胞HeLa细胞,我们将深入探究IGF-1调节上述经典NF-κB和NLRP3炎症通路激活的上游分子生物学机制,同时力求发现IGF-1调节癌细胞发生炎症的通路关键蛋白并找到其抑制剂。构建IRS-1原核表达载体及利用Pull-down技术对IRS-1的下游蛋白进行挖掘;利用Western blot方法对IGF-1通过IRS-1诱导HeLa细胞中COX2产量进行观察;利用免疫共沉淀技术检测IGF-1通过COX2诱导HeLa细胞中mPGES-1含量,并对mPGES-1与p-ERK的关系进行考察;利用Western blot技术分别检测抑制剂PD98059、SB203580、SP600125预处理IGF-1诱导细胞的ERK、JNK、p38蛋白的磷酸化水平,并分析IGF-1诱导后HeLa细胞中p-p38与RAC2以及RAC2与NOX2蛋白表达之间的关系;利用免疫荧光技术和Western blot技术对关键通路蛋白敲低细胞株进行验证;利用荧光酶标仪、ELISA和Western blot等方法对IRS-1、mPGES-1和NOX2敲低细胞株和抑制剂作用后的HeLa细胞中ROS、炎性因子IL-1β、IL-6和TNF-α以及炎症通路蛋白IKK、NF-κB、NLRP3、IL-1β、ASC和Caspase 1的表达进行检测。结果表明,带有GST标签的IRS-1蛋白原核表达成功;Pull-down实验结果证明IGF-1能够通过IRS-1介导COX2的表达,免疫共沉淀实验表明IGF-1能够通过COX2诱导mPGES-1的表达,mPGES-1抑制剂MF63处理结果表明mPGES-1调控ERK表达;并且,MAPKs通路抑制剂处理发现ERK位于JNK和p-38上游,JNK位于p-38的上游;这些结果表明IRS-1/COX2/mPGES-1能够调节MAPKs(ERK、JNK和p-38)表达;免疫共沉淀和Western blot实验证明,磷酸化的p-38能够通过RAC2调节NOX2的表达;IRS-1抑制剂NT157、COX2抑制剂Celecoxib、mPGES-1抑制剂MF63、MAPKs抑制剂(PD98059、SB203580和SP600125)、RAC2抑制剂NSC23766和NOX2抑制剂DPI能够抑制IGF-1诱导HeLa细胞产生的ROS、炎性因子IL-1β、IL-6和TNF-α以及炎症通路蛋白IKK、NF-κB、NLRP3、IL-1β、ASC和Caspase 1的表达量,这表明IRS-1/COX2/mPGES-1/MAPKs/RAC2/NOX2调节NF-κB和NLRP3的表达;另外,我们成功构建IRS-1、mPGES-1和NOX2三种蛋白敲低细胞株,并且发现IGF-1能够诱导IRS-1、mPGES-1和NOX2敲低HeLa细胞株产生的ROS、炎性因子IL-1β、IL-6和TNF-α以及炎症通路蛋白IKK、NF-κB、NLRP3、IL-1β、ASC和Caspase1的表达量低于未敲除HeLa细胞株,这些结果确证IRS-1/mPGES-1/NOX2能够调节NF-κB和NLRP3的表达。综上所述,我们在国内外率先发现IRS-1/mPGES-1/NOX2的表达能够激活经典NF-κB和NLRP3炎症通路,并且IGF-1通过上述机制调节癌细胞炎症的发生,同时我们还发现IRS-1、mPGES-1、NOX2为IGF-1调节癌细胞发生炎症通路的关键蛋白,其各自抑制剂组合NT157+MF63+DPI能够显著抑制体外癌细胞炎症发生。最后,我们建立B16-F10细胞肺荷瘤鼠模型,在体内验证IGF-1诱导癌细胞发生炎症反应的机制,考查炎症反应对癌细胞组织浸润和血管生成能力的影响,以及IRS-1、mPGES-1、NOX2抑制剂组合(NT157+MF63+DPI)作为抑制癌症炎症的候选药物的可能性。利用组织病理切片和HE染色对肺荷瘤鼠肺部肿瘤和其他主要脏器进行观察;利用Western blot和ELISA方法对B16-F10细胞肺荷瘤鼠体内炎性因子IL-1β、IL-6、TNF-α以及炎症通路蛋白IKK、NF-κB、NLRP3、IL-1β、ASC和Caspase1的表达以及通路蛋白IRS-1/COX2/mPGES-1/MAPKs/RAC2/NOX2的含量进行检测。结果表明,IGF-1诱导组荷瘤鼠体重较荷瘤模型组显著降低,而IRS-1、mPGES-1、NOX2抑制剂组合处理组荷瘤鼠体重较正常组差异不显著,较荷瘤模型组和IGF-1诱导组体重显著增加;病理组织学结果表明,IGF-1显著诱导荷瘤鼠肿瘤结节,抑制剂组合处理组显著抑制肿瘤结节数量,抑瘤率为31.81%;IGF-1显著诱导肺部肿瘤血管生成和组织浸润性,而抑制剂组合能够减轻IGF-1诱导组的上述变化;抑制剂组合处理组荷瘤鼠外周血中的炎性因子IL-1β、IL-6和TNF-α以及组织中炎症通路蛋白IKK、NF-κB、NLRP3、IL-1β、ASC和Caspase 1的表达显著低于荷瘤模型组和IGF-1诱导组;抑制剂组合处理组荷瘤鼠肿瘤组织中通路蛋白IRS-1、COX2、mPGES-1、p-ERK、p-JNK、p-p38、RAC、NOX2的表达量显著低于荷瘤模型组和IGF-1诱导组。综上所述,IGF-1能够加重体内肿瘤炎症反应,该炎症反应由经典的NF-κB和NLRP3通路介导,该通路上游由IRS-1/mPGES-1/NOX2调节,IRS-1、mPGES-1和NOX2抑制剂组合可以作为抑制癌症炎症的候选药物,该结果与体外实验相一致。总之,本研究结果表明,我们率先发现IGF-1能够诱导癌症炎症的发生,并且该炎症反应由经典的NF-κB和NLRP3通路介导,特别是,我们首次证明该经典炎症通路激活是由上游的IRS-1/mPGES-1/NOX2调节,本研究的亮点是发现癌症炎症激活通路关键蛋白IRS-1、mPGES-1和NOX2的抑制剂组合能够显著抑制癌细胞炎症的发生、组织浸润和血管生成,这为人和动物抗癌新药研发提供了重要物质基础和理论基础,也为癌症病人和动物的带瘤生存提供了可能。更多还原

【Abstract】 With the rapid economic growth,people’s living habits and living environment have undergone tremendous changes,followed with various diseases bringing harm to human beings.The occurrence and development of cancers account for a large proportion among these diseases.There were approximately 18.08 million new cases of cancer and 9.56 million cancer deaths worldwide in 2018.At the same time,about20% ~ 30% of pet dogs and cats die each year from cancer caused by various causes,resulting in economic losses of up to 10 billion yuan.There are hundreds types of cancer in animals and humans reported currently.and they all have similar common characteristics.Among them,the inflammation response caused by cancer is the main cause of cancer cachexia and recurrence.Cancer-related inflammation can promote the generation of new blood vessels and cancer cells metastasize.Inflammatory response is a series of complex biological responses produced by the body in order to destroy foreign bodies from inside and outside the body.Reactive oxygen species(ROS),a cell by-product during normal metabolism,plays an important role in the development of inflammation and cancer,and successfully links inflammation to cancer.At the same time,extracellular stimulation could also lead to an increase of intracellular ROS production,leading to a series of biological reactions.NF-κB is an oxidative stress sensor that regulates a variety of cellular functions,including inflammatory signaling,and abnormal activation of NF-κB is related to the occurrence and development of cancer.The NLRP3 inflammatory body is an important inflammatory response regulator.When activated,it will produce an inflammatory cascade and can promote the development and metastasis of cancer.As a growth factor analog,insulin-like growth factor 1(IGF-1)regulates the expression of inflammatory factors and is involved in the development of various cancers,but its mechanism of action has not been reported.In this study,in the consideration of the role of IGF-1 in inflammatory response and cancer,we used human cervical cancer cell(HeLa),Canine osteosarcoma cell(D-17)and mouse melanoma cell(B16-F10)compared with human,dog and mouse normalcells(NCM-460 cells,MDCK cells and MODE-K cells)in vitro,and combined with in vivo tumor experiments to explore whether IGF-1 can activate classical NF-κB and NLRP3 pathway mediate the inflammatory response of cancer cells.The most important thing is that we will first explore the upstream molecular mechanism of IGF-1 regulating NF-κB and NLRP3 inflammation pathway activation at home and abroad,so as to discover the key proteins that IGF-1 regulate the inflammation pathway of cancer cells and find their inhibitors.Finally provide solutions to suppress the occurrence of cancer-related inflammation,in order to achieve cancer patients and animals with tumor survival.First,we investigated whether IGF-1 can induce inflammatory phenotypes in human,canine,and mouse cancer cells(HeLa cells,D-17 cells,and B16-F10 cells).The fluorescence microplate reader was used to observe the changes in the production of ROS produced by IGF-1 induced the three kinds of cancer cells.qPCR and ELISA methods were used to detect the expression of inflammatory factors IL-1β,IL-6 and TNF-α of the three kinds of cancer cells and normal cells of human,dog and mouse(NCM-460 cells,MDCK cells,and MODE-K cells)induced by IGF-1.The results show that IGF-1 can induce ROS production in the cytoplasm of HeLa cells,D-17 cells and B16-F10 cells,and the optimal induction concentration is 100 ng/mL.The best induction time was 120 minutes.Mitochondria ROS inhibitor(MitoQ)had no effect on IGF-1 induced ROS production in the three kinds of cancer cells,while NOX2 inhibitor(DPI)significantly inhibited IGF-1 induces ROS production in the three kinds of cancer cells,which indicates that IGF-1 induces ROS produced by HeLa cells,D-17 cells and B16-F10 cells mainly from the cytoplasm and is affected by NOX2 mediated.IGF-1 can induce the increase of inflammatory factors IL-1β,IL-6 and TNF-α secreted and their mRNA expression by the three kinds of cancer cells.ROS scavenger(NAC)can significantly inhibited IGF-1-induced increase of mRNA levels of IL-1β,IL-6 and TNF-α in the three kinds of cancer cells,as well as the expression of three inflammatory factors in the cell supernatant.And we found that under the same treatment conditions,IGF-1 could not induce NCM-460 cells,MDCK cells,and MODE-K cells of human,dog,and mouse to produce increased ROS production and increased expression of the above-mentioned inflammatory cytokines.In summary,IGF-1 can only induce the inflammatory response phenotype in human and animal cancer cells,but cannot induce the inflammatory response phenotype of normal cells,so it is worth further studying the mechanism of IGF-1mediated cancer cell inflammatory response.Secondly,for human,canine and mouse cancer cells,and using normal cells as controls,we explored whether IGF-1 mediates the inflammatory response of cancer cells by activating the classic NF-κB and NLRP3 pathways.Western blot was used to detect the expression of NF-κB and NLRP3-related inflammatory pathway proteins in HeLa cells,D-17 cells,B16-F10 cells and normal cells(NCM-460 cells,MDCK cells and MODE-K cells)of human,dog and mouse after IGF-1 treatment.qPCR and ELISA were used to detect the expression of IL-1β,IL-6 and TNF-α in HeLa cells,D-17 cells and B16-F10 cells under the action of NF-κB inhibitor BAY 11-7082 and Capase 1 inhibitor Ac-YVAD-cmk.The results show that IGF-1 can induce the high expression of the inflammatory pathway proteins IKK,NF-κB,NLRP3,IL-1β,ASC and Caspase 1 in three types of human,dog and mouse cancer cells,and the ROS scavenger NAC can significantly inhibit the expression of IKK,NF-κB,NLRP3,IL-1β,ASC and Caspase 1.NF-κB inhibitor and Caspase 1 inhibitor can significantly inhibit IGF-1-induced the mRNA expression of IL-1β,IL-6 and TNF-α in three cancer cells and the secretion of three inflammatory factors in the cell supernatant.Under the same treatment conditions as the three cancer cells,IGF-1 could not induce changes in the expression of NF-κB and NLRP3-related inflammatory pathway proteins in normal cells.Taken together,the results indicate that IGF-1 can induce the expression of NF-κB and NLRP3-related inflammatory pathway proteins in cancer cells of humans,dogs and mice,and cannot activate the expression of corresponding pathway proteins in normal cells.In summary,IGF-1 can mediate the inflammatory response of cancer cells by activating the classic NF-κB and NLRP3 pathways.Thirdly,for HeLa cells as cancer model cell,we will deeply investigate the upstream molecular biological mechanism of IGF-1 regulating the above-mentioned classic NF-κB and NLRP3 inflammation pathway activation.At the same time,we strive to find key proteins and inhibitors of IGF-1 regulating the inflammation pathway of cancer cells and its inhibitor.Construction of IRS-1 prokaryotic expression vector and mining of downstream proteins of IRS-1 by Pull-down technology.Western blot was used to observe the IGF-1 induced COX2 production in HeLa cells by IRS-1.The content of mPGES-1 in HeLa cells induced by IGF-1 via COX2 and the protein-protein interactions(p-p38?RAC2)were detected by co-immunoprecipitation.Western blot was used to detect the phosphorylation levels of ERK,JNK and p38 proteins induced by IGF-1 pretreated with PD98059,XSB203580 and SP600125,and analysis of the relationship between p-p38?RAC2 and RAC2?NOX2 expression in HeLa cells induced by IGF-1.The key pathway protein knockdown cell line was verified by immunofluorescence and Western blot.ROS,inflammatory factors IL-1β,IL-6,and TNF-α,and inflammatory pathway proteins IKK,NF-κB,NLRP3,IL-1β,ASC and Caspase 1 in HeLa cells,IRS-1,mPGES-1 and NOX2 knockdown cell lines and treated with inhibitors were detected using fluorescence microplate reader,ELISA and Western blot.The results showed that the GRS-tagged IRS-1 protein was successfully expressed prokaryotically.The results of the Pull-down experiments proved that IGF-1 can mediate the expression of COX2 through IRS-1,and co-immunoprecipitation experiments showed that IGF-1 can induce the expression of mPGES-1 through COX2.mPGES-1 inhibitor MF63 treatment showed that mPGES-1 regulates ERK expression,and MAPKs pathway inhibitors treatment revealed that ERK is located upstream of JNK and p-38,and JNK is located upstream of p-38.These results indicate that IRS-1/COX2/mPGES-1 can regulate the expression of MAPKs(ERK,JNK and p-38).Co-immunoprecipitation and Western blot experiments show that phosphorylated p-38 can regulate the expression of NOX2 through RAC2.IRS-1 inhibitor NT157,COX2 inhibitor Celecoxib,mPGES-1 inhibitor MF63,MAPKs inhibitor(PD98059,SB203580,and SP600125),RAC2 inhibitor NSC23766 and NOX2 inhibitor DPI can inhibit IGF-1induced ROS,inflammatory factors IL-1β,IL-6 and TNF-α,and inflammatory pathway proteins IKK,NF-κB,NLRP3,IL-1β,ASC and Caspase 1 expression levels produced by HeLa cells,which indicates that IRS-1/COX2/mPGES-1/MAPKs/RAC2/NOX2 regulates the expression of NF-κB and NLRP3.In addition,we successfully constructed IRS-1,mPGES-1 and NOX2 knockdown cell lines,and IGF-1 was found to induce IRS-1,mPGES-1 and NOX2 knockdown HeLa cells the expression levels of ROS,inflammatory factors IL-1β,IL-6 and TNF-α and the inflammatory pathway proteins IKK,NF-κB,NLRP3,IL-1β,ASC and Caspase 1 were lower than those of non-knocked HeLa cell lines.These results confirm that IRS-1/mPGES-1/NOX2 can regulate the expression of NF-κB and NLRP3.In summary,we are the first to discover that the expression of IRS-1/mPGES-1/NOX2 can activate the classic NF-κB and NLRP3 inflammation pathways at home and abroad,and IGF-1 regulates the occurrence of cancer cell inflammation through the above mechanism.It was found that IRS-1,mPGES-1 and NOX2 are the key proteins that IGF-1 regulates the inflammatory pathway of cancer cells,and their respective inhibitor combinations NT157 + MF63 + DPI can significantly inhibit the development of cancer cells in vitro.Finally,we established a B16-F10 cell lung metastasis-bearing mouse model to verify the mechanism by which IGF-1 induces inflammatory responses in cancer cells in vivo and to examine the effects of inflammatory response on cancer cell metastasis and angiogenesis and the possibility of IRS-1,mPGES-1 and NOX2 inhibitor combination(NT157 + MF63 + DPI)as a candidate drug to inhibit cancer inflammation.Histopathological sections and HE staining were used to observe lung tumors and other major organs in lung metastatic tumor-bearing mice.Western blot and ELISA were used to detect the expressions of inflammatory factors IL-1β,IL-6,TNF-α and inflammatory pathway proteins IKK,NF-κB,NLRP3,IL-1β,ASC and Caspase 1 and the contents of pathway proteins IRS-1/COX2/mPGES-1/MAPKs/RAC2/NOX2.The results showed that the body weight of tumor-bearing mice in the IGF-1 induction group was significantly lower than that in the tumor-bearing model group,while the weight of tumor-bearing mice in the IRS-1,mPGES-1,and NOX2 inhibitor combination treatment group was not significantly different from that in the normal group,and compared with the tumor-bearing model group and IGF-1 induction group significantly increased weight.Pathological histological results showed that IGF-1 significantly induced tumor nodules in tumor-bearing mice,inhibitors combination treatment group significantly inhibited the number of tumor nodules,and the tumor inhibition rate was 31.81%.IGF-1 induce lung tumor angiogenesis and tissue infiltration significantly,and the inhibitor combination can reduce the above changes in the IGF-1 induction group.The expressions of inflammatory factors IL-1β,IL-6,TNF-α in the peripheral blood and inflammatory pathway proteins IKK,NF-κB,NLRP3,IL-1β,ASC and Caspase 1 in tissues of inhibitor combination treatment group were significantly lower than those in the tumor-bearing model group and the IGF-1 induction group.The expressions of pathway proteins IRS-1,COX2,mPGES-1,p-ERK,p-JNK,p-p38,RAC,and NOX2 in tumor tissues of inhibitor combination treatment group were significantly lower than those in the tumor-bearing model group and the IGF-1 induction group.In summary,IGF-1 can aggravate tumor inflammatory responses in vivo.The inflammatory response is mediated by the classic NF-κB and NLRP3 pathways,which are upstream regulated by IRS-1/mPGES-1/NOX2.The combination of IRS-1,mPGES-1 and NOX2 inhibitors can be used as candidate drugs to inhibit cancer inflammation,and the results are consistent with in vitroexperiments.In conclusion,the results of this study indicate that we were the first to discover that IGF-1 can induce cancer inflammation,and that the inflammatory response is mediated by the classic NF-κB and NLRP3 pathways.In particular,we have shown for the first time that the activation of this classic inflammatory pathway is regulated by upstream IRS-1/mPGES-1/NOX2.The highlight of this study was to discover that the cancer inflammation activation pathway key proteins IRS-1,mPGES-1 and NOX2 inhibitors can significantly inhibit the development of cancer cell inflammation,tissue infiltration and blood vessels generation,which provides an important material and theoretical basis for the development of new anti-cancer drugs for humans and animals,as well as the possibility of tumor-bearing survival for humans and animals.更多还原