Liuwei Dihuang Pill Inhibits N-methyl-N-nitrosourea-induced Gastric Tumorigenesis In Db/db Mice

Aim: In in vivo study,We aimed to explore the effects of Liuwei Dihuang Pill(LDP)on N-methyl-N-nitrosourea-induced gastric tumorigenesis and gastric mucosal proliferation.We investigated the effects of LDP on insulin/insulin-like growth factor(IGF)-1 signaling,serum and gastric tissue IL-6,TNF-α levels,serum adiponectin and leptin to elucidate the antitumor mechanisms of LDP.In in vitro study,We detected the influences of LDP on gastric cancer cell MKN45 proliferation,apoptosis,cell cycle and IGF-1/IGF-1R signaling pathway.We expect to provide experimental evidence for prevention of type 2 diabetes-related gastric cancer by LDP.Methods: In in vivo study,all mice were divided into 4 groups randomly,db/m mice as the nondiabetic control,db/db as the diabetic control,metformin-treated db/db mice as the positive control,LDP-treated db/db mice as the treatment group.All mice drank MNU solution for 20 k to induce gastric cancer,LDP and metformin were used for 10 k.The body weight and food intake were measured once a week.At weeks 1,20 and 30,all mice were subject to fasting blood glucose.The incidence of gastric cancer and dysplasia was detected with HE staining,and gastric mucosal proliferation was measured by immunohistochemistry staining for Ki67.Serum insulin,IGF-1,IL-6,TNF-α,adiponectin,leptin were detected with commercial Elisa kits.HOMA-IR was calculated with fasting blood glucose and insulin.IGF-1R/AKT and their phosphorylated proteins were examined with western blotting.IGF-1,IGF-1 receptor(IGF-1R),IGF-2,IGF-2 receptor(IGF-2R),insulin receptor,IL-6,TNF-α mRNAs were detected with qRT-PCR.In in vitro study,the effects of LDP on gastric cell MKN45 proliferation,apoptosis,and cell cycle were examined by CCK-8,Annexin V-FITC/PI,and flow cytometry,respectively.The phosphorylated proteins of IGF-1R,AKT,ERK1/2,JNK,P38 and PARP protein were detected with western blotting.After treatment with IGF-1and LDP,we repeated the above experiment to determinedwhether LDP exerted inhibitory effects on IGF-1induced proliferation,apoptosis inhibition,cell cycle progression and activated IGF-1R signaling.Results: In in vivo study,the incidence of gastric dysplasia was significantly elevated in diabetic control compared with nondiabetic control,while was significantly decreased by LDP and metformin.Two gastric cancer was found in diabetic control,while no gastric cancer was found in other three group.The gastric mucosal proliferation was significantly increased in diabetic control,and was markedly decreased in LDP or metformin treated group,as shown by decrease in percentage of Ki67 positive cells.Compared with diabetic control,LDP significantly decreased HOMA-IR,serum hyperglycemia,hyperinsulinemia,high IGF-1,and also markedly downregulated expression of IGF-1,insulin receptor mRNAs,and phosphor-IGF-1R,phosphor-AKT proteins,but did not influenced IGF-2,IGF-2R mRNAs,and total IGF-1R,AKT proteins.In addition,serum IL-6,TNF-α and gastric IL-6 mRNA were significantly reduced by LDP,but gastric TNF-α mRNA was not decreased by LDP.LDP treated mice had higher adiponectin and lower leptin than diabetic control.Similar results were found in metformin treated group.In in vitro study,8 mg/ml LDP did not inhibit gastric cancer cell,and higher concentration of LDP(12,16 mg/ml)has significantly inhibitory effect on gastric cancer cell.After treated with 16 mg/ml LDP for 24 h,gastric cancer cell proliferation was decreased in time-dependent way.16mg/ml LDP exerted inhibitory effect on IGF-1 induced gastric cancer cell proliferation.In addition,gastric cancer cell apoptosis and cleaved PARP was greatly induced by 16mg/ml LDP.Moreover,the inhibitory effect of IGF-1 on gastric cancer cell apoptosis was reversed by 16 mg/ml LDP.LDP also induced cell cycle arrest as demonstrated by increased proportion of S cells and decreased proportion of G2 cells.Furthermore,LDP downregulated expression of phosphorylated IGF-1R/AKT proteins induced by IGF-1,while did not alter expression of phosphorylated MAPK proteins(ERK1/2,P38,and JNK)induced by IGF-1.Conclusion: in vivo study demonstrated that LDP inhibited the increased risk of MNU-induced gastric tumorigenesis in type 2 diabetes by suppressing gastric mucosal proliferation,which was similar to metformin.The antitumor mechanisms of LDP may be associated with alleviating insulin resistance,which led to decreasedinsulin/IGF-1/IGF-1R/AKT signaling,and reducing proinflammatory cytokines in serum and gastric tissue.The increase of adiponectin and decrease of leptin may also contribute to the antitumor effect of LDP.The antitumor mechanisms are at least in part similar to metformin.Moreover,in vitro study demonstrated that LDP inhibited gastric cancer cell proliferation,and induced apoptosis,cell cycle arrest by IGF-1/IGF-1R/AKT signaling rather than MAPK signaling.