Analysis Of Intestinal Microbiota Before And After Transcatheter Arterial Chemoembolization In Patients With Primary Liver Cancer And The Regulatory Role And Mechanism Of Nicotinamide Single Nucleotide(NMN) In Hepatocellular Carcinoma

Objective: Hepatocellular carcinoma(HCC)is the most common primary liver cancer,ranking fifth in the world.there are few available treatment options for advanced HCC.Even with active treatment,such as liver transplantation,resection,percutaneous ablation and transcatheter arterial chemoembolization,HCC may still recur and metastasize,and the 5-year survival rate is less than 20%.In addition,traditional chemotherapy and molecular targeted drugs are hindered by tumor heter ogeneity and inherent and acquired drug resistance that may occur in tumors.The se characteristics limit the efficacy of systemic therapy for HCC patients.Therefore,it is urgent to study new strategies to improve the clinical outcomes of HCC patients.As a "virtual metabolic organ",intestinal flora forms an axis with kidney,brain,cardiovascular system,skeletal system and other extraintestinal organs.In recent years,the intestinal-hepatic axis has attracted more and more attention.Intestinal-hepatic axis is the result of close interaction between gastrointestinal tract and liver in anatomy and function,mainly through portal vein circulation.The symbiotic relationship between intestinal flora and liver is stably regulated by a complex in teraction network,which includes the interaction between metabolism,immunity and neuroendocrine.Intestinal axis affects the pathogenesis of liver diseases,which is an important focus of clinical research.It is reported that the hepatic-intestinal axis plays a key role in the pathogenesis of hepatocellular carcinoma(HCC),but the influence of intestinal flora on the clinical outcome of HCC patients is still unclear,and the relationship between intestinal flora and HCC remains to be clarified.Metabonomics is a top-down platform in the field of systems biology,which mainly focuses on the dynamic changes of the interference of small molecules on organisms.Non-targeted metabolomics methods are widely used to discover new biomarkers and study carcinogenic mechanisms.These methods are also used to study chronic liver disease(CLD)and hepatocellular carcinoma(HCC).It has been reported that some serum or urine metabolites,such as sphingosine,bile acid,glycine,trimethylamine-N-oxide and dipeptide,are differential metabolites or candi date biomarkers of CLD or HCC.Serum,urine and feces are generally regarded as metabolic pools reflecting the metabolic disorder of patients,and the markers in these biological fluids can reflect the characteristics of the system during the disease process.Nicotinamide(NAM)is an amide form of semi-essential vitamin B3.Many foods,including meat,fish,eggs,beans,mushrooms,nuts and grains contain nicotinamide.NAM is synthesized de novo through tryptophan metabolism,and is easily absorbed by gastrointestinal tract after ingestion,and widely distributed in body tissues.Nicotinamide mononucleotide(NMN)can be directly synthesized by ATP catalyzed by Nicotinamide mononucleotide adenosine transferase(NMNAT).Nicot inamide adenine dinucleotide(NAD+),NAD+ is a large number of metabolites that can be enriched in the body and has the function of maintaining cell homeostasis.It is reported that nicotinamide can prevent skin cancer,but the development process of liver cancer is still unclear.This study mainly discusses the role and mechanism of nicotinamide mononucleotide(NMN)in liver cancer,and provides the basis for developing new treatment strategies for liver cancer.Methods: Part I: The changes of intestinal flora and metabolites in patients with primary liver cancer before and after percutaneous hepatic arteriography(HAG)combined with transcatheter arterial chemoembolization(TACE)were preliminarily studied.Firstly,the stool samples of 30 patients with primary liver cancer before and after percutaneous hepatic arteriography(HAG)and transcatheter arterial chemoe mbolization(TACE)were used to sequence the 16 Sr DNA of V3V4 region,and the α diversity,β diversity and significant difference of LEf Se were analyzed by QIIME2 dada2 and Vsearch software to evaluate the changes of intestinal flora structure of patients with primary liver cancer before and after operation.Secondly,through extensive targeted metabolomics detection of stool samples from 30 patients with liver cancer before and after operation,mass spectrometry data was processed by software Analyst 1.6.3,and qualitative and quantitative analysis of metabolites,sample quality control analysis,PCA and cluster analysis were carried out.The changes of metabolic pathways of stool samples from patients with liver cancer before and after operation were compared,and the differential metabolic pathways were selected for follow-up study.Part II: To further explore the role and mechanism of nicotinamide mononucl eotide(NMN)in hepatocellular carcinoma through cell experiment.Firstly,low,medium and high doses of amide mononucleotide(NMN)were used to intervene he patoma cells Hep G2 and HH-7,and the NAD+ concentration and NAD+/NADH r atio in the cells were detected by the kit;Cell proliferation and apoptosis were detected by CCK-8 and Hoechst staining.The expression of apoptosis-related protein Bax,Bcl-2,cleaved PARP and caspase-3 activity,caspase-9 activity in Hep-G2 and Huh7 was detected,and the effect of nicotinamide mononucleotide(NMN)on the proliferation and apoptosis of hepatoma cells was discussed.In order to study the effect of nicotinamide mononucleotide(NMN)on the autophagy of hepatoma cells,The expression of LC3B(LC3B-II/LC3B-I ratio),Beclin 1,ATG5 and p62 were detected by Western blot;In order to study the effect of nicotinamide mo nonucleotide(NMN)on ferroptosis in hepatoma cells,after different concentrations of NMN intervened the cells,the intracellular ROS level,MDA content,SOD activity,GSH level and intracellular iron ion level were detected by using DCFH-D A fluorescent probe;The levels of ACSL4,SLC7A11 and GPX4 were detected by Real-time PCR and Western blot;In order to further study the effect of nicotina mide mononucleotide(NMN)on AMPK/m TOR signaling pathway,after different concentrations of NMN intervened the cells,the AMPK,p-AMPK,m TOR,p-m TO R,p70S6 K and p-p70S6 Kin the cells were detected by estern blot.Knock down A MPK to explore the anticancer effect of nicotinamide mononucleotide(NMN),and verify the recovery experiment.After AMPK siRNA transfected the cells,and H-NMN(high dose NMN)treated the cells for 48 h,Western blot tested the autophagy-related single markers LC3B(LC3B-II/LC3B-I ratio was calculated),p62,ferr optosis-related proteins SLC7A11 and GPX4 were detected Hoechst staining was used to detect apoptosis;The reagent kit was used to detect intracellular ROS level,MDA content,SOD activity,GSH level and intracellular iron ion level.Part III:the effect of nicotinamide mononucleotide(NMN)on human hepatoce llular carcinoma xenograft was verified at animal level.Hep G2 cells were cultured and subcutaneously injected into the left armpit to establish a nude mouse model of human liver cancer cell line.The low,middle and high dose groups of NMN were administered with 6 rats in each group.Three days after cell injection,NMN was administered orally to each group once a day for three weeks.The NAD+ concentration of liver tumor tissues were detected with the kit.HE staining was used to detect the tumor pathological morphology in the tumor tissues of nude mice.was used to detect the expression and distribution of Ki-67 and PCNA prot eins expressing was tested with Immunohistochemistry.TUNEL staining detected the apoptosis of the hepatoma cell.Results: Part I: Firstly,we performed 16 S rDNA high-throughput sequencing on the stool samples of 30 patients with primary liver cancer before and after operation,and spliced the paired Reads obtained by double-ended sequencing into a sequence by QIIME2 dada2 analysis software.Then,the spliced Reads were subjected to primer removal,splicing,quality filtering,weight removal,chimerism removal,clustering and other steps.A total of 4,312,319 effective sequences were obtained,with 99% of the average sequence length ranging from 400 BP to 430 BP.At 96% similarity,cluster analysis was carried out by using Vsearch software,and chimera filtering was carried out on the clustered sequences to obtain OTUs for species classification,with each OTU representing a species.A total of 10,856 OTUs were obtained in this study.Through α diversity analysis,it was found that the in dexes of Chao1,observed species and Faith_pd in EG group before operation were higher than those in CG group after operation,which revealed that the intestinal microbial diversity of patients with liver cancer after operation decreased.Through β diversity analysis,it was found that there were significant differences in β diversity of intestinal flora between CG group and EG group.LEf Se analysis(LD A Effect Size)was used to evaluate the influence of species abundance on the differences between groups,and to find out the bacteria with significant contribution in the two groups of samples of EG before operation and CG after operation.The LDA scores of bacteria with significant differences in different groups were an alyzed and counted.It was found that Rhodobacter,Rhodobacter,Microbacteriaceae,Moraxella,Bacteroidaceae,Porphyromonadaceae,The distribution of Bacteroides,Bacteroides,Macellibacteroides and Haldeman in EG group was significantly higher than that in CG group.Secondly,Through the screening of metabonomics differ ence metabolites in stool samples of 30 patients with liver cancer before and after TACE(EG),74 different metabolites were found in CG group,of which 24 met abolites were down-regulated and 50 metabolites were up-regulated,among which NAD+ was significantly up-regulated after TACE.According to the path analysis of KEGG database,eight main metabolic pathways were found: phospholipid bios ynthesis,nucleotide sugar metabolism,nicotinic acid and nicotinamide metabolism,mitochondrial electron transfer chain,galactose Galactosemiall metabolism,galactose galactose miall metabolism,middle galactose metabolism and cardiac phospholipid biosynthesis.Combined analysis of microbiology and metabolomics found that there were significant differences between the changes of intestinal flora before and after operation and the metabolism of nicotinic acid and nicotinamide.Transcatheter arterial chemoembolization(TACE)in patients with primary liver cancer leads to the change of intestinal flora,which may be due to the decrease of some harmful bacteria such as Moraxella and Haldeman,which activate nicotinic acid and nicotinamide metabolic pathways,increase NAD+ and then play an anti-cancer role.At present,the specific role and regulatory mechanism of nicotinic acid and nicotinamide metabolism in hepatocellular carcinoma are still unclear,so we choose nicotinic acid and nicotinamide metabolic pathway for the follow-up basic research,and choose nicotinamide mononucleotide NMN,the intermediate of nicotinic acid and nicotinamide metabolic pathway,to study the role and mechanism of NMN in hepatocellular carcinoma HCC.Part II: To verify the role and mechanism of nicotinamide mononucleotide(NMN)in hepatocellular carcinoma at cellular level.The kit detected the concentration of NAD+ and found that NMN could up-regulate the concentration of NAD+and the ratio of NAD+/NADH in Hep-G2 and Huh7 cells;CCK-8 results showed that NMN could inhibit the proliferation of Hep-G2 and Huh7 cells.Hoechst staining showed that NMN could promote the apoptosis of Hep-G2 and Huh7 cells.The results of Western Blot showed that NMN increased the expression of apopto sis-related proteins Bax and cleaved PARP,and decreased the expression of Bcl-2protein.Caspase-3 and caspase-9 activity kits showed that NMN could promote the activities of caspase-3 and caspase-9 in Hep-G2 and Huh7 cells;In order to verify the effect of nicotinamide mononucleotide(NMN)on the autophagy of hepatoma cells,the expression of LC3 B,Beclin 1,ATG5 and p62 was detected.the re sults showed that NMN could increased the ratio of LC3B-II/LC3B-I between He p-G2 and Huh7 cells,increased the expression of Beclin 1 and ATG5 proteins,and decreased the protein expression of p62;In order to verify the effect of nicotinamide mononucleotide(NMN)on iron death of hepatoma cells,the levels of RO S,MDA,SOD,GSH and intracellular iron ions were detected.The results showed that NMN up-regulated ROS and MDA levels in Hep-G2 and Huh7 hepatoma cells,reduced SOD activity and GSH levels,and increased iron ion levels.The proteins expression of ACSL4,SLC7A11 and GPX4 were detected by Real-time PCR and WB.The results showed that NMN upregulated the expression of ACSL4 protein and down-regulated the expression of SLC7A11 and GPX4 protein in Hep-G2 and Huh7 hepatoma cells;To verify the effect of nicotinamide mononucleotid e(NMN)on AMPK/mTOR signaling pathway,Western blot deteced the proteins expression level of AMPK,p-AMPK,m TOR,p-m TOR,p70S6 K and p-p70S6 K in hepatocellular carcinoma cells.Results NMN can promoted the expression of p-AMPK and reduced the expression of p-m TOR and p-p70S6 Kin Hep-G2 and Huh7cells;In order to further verify the role of NMN through AMPK pathway,the expression of LC3 B,p62,SLC7A11 and GPX4 in cells was detected by knock-down AMPK(AMPK si RNA),the apoptosis was detected by Hoechst staining,the R OS level,MDA content,SOD activity,GSH level and intracellular iron ion level were detected by kit,and the recovery experiment was carried out.Results showed that AMPK si RNA reversed the acceleration of LC3B-II/LC3B-I ratio,Beclin 1and ATG5 protein in Hep-G2 and Huh7 cells regulated by NMN,and improved the apoptosis and ROS level,MDA content,SOD activity,GSH level and intracell ular iron ion level regulated by NMN.Part III:To verify the role of nicotinamide mononucleotide(NMN)in liver cancer at animal level: NMN can inhibit the growth of transplanted tumor of human liver cancer cell line in nude mice,and up-regulate NAD+ concentration and N AD+/NADH ratio in tumor tissue;The results of HE showed that the cell necrosis in tumor tissues of NMN middle dose group and NMN high dose group increased gradually,and the cell necrosis in tumor tissues of NMN high dose group was the most serious.IHC detected the distribution of Ki-67 and PCNA of tumor tissue.The results showed that protein expression of Ki-67 and PCNA in the NMN high dose group decreased.TUNEL staining was used to detect the apoptosis of tumor tissues.It was found that nicotinamide mononucleotide(NMN)could pr omote the apoptosis of tumor tissues in nude mice transplanted with human hepat ocellular carcinoma cell line.Conclusion: 1.The analysis of intestinal flora shows that the diversity of intestinal microorganisms in patients with liver cancer after operation is reduced;Through the analysis of β diversity,it was found that there were significant differences in the β diversity of intestinal flora between preoperative patients with liver cancer(CG group)and postoperative patients with liver cancer(EG group).Bacteroid es and Parabacteroides,the dominant and beneficial flora in intestinal flora of patients with liver cancer,decreased significantly after operation.2.Metabonomics analysis showed that there were 74 different metabolites in CG group compared with EG group,24 metabolites were down-regulated and 50 metabolites were up-regulated,among which NAD+ was significantly up-regulated.According to the path analysis of KEGG database,eight major metabolic pathways were found: phospholipid biosynthesis,nucleotide sugar metabolism,nicotinic acid and nicotinamide metabolism,mitochondrial electron transfer chain,galactose galactose Galactosemiall metabolism,galactose galactose metabolism,and cardiac phospholipid biosynthesis.Bioinformatics is used to analyze the metabolic characteristics of liver cancer.Combined analysis of microbiology and metabolomics found that there were significant differences between the changes of intestinal flora before and after operation and the metabolism of nicotinic acid and nicotinamide.Tran scatheter arterial chemoembolization(TACE)in patients with primary liver cancer leads to the change of intestinal flora,which may be due to the decrease of some harmful bacteria such as Moraxella and Haldeman,which activate nicotinic acid and nicotinamide metabolic pathways,increase NAD+ and then play an anti-cancer role.At present,the specific role and regulation mechanism of nicotinic acid and nicotinamide metabolism in hepatocellular carcinoma are still unclear,so we choose nicotinic acid and nicotinamide metabolism pathway for the follow-up basic research,and nicotinamide mononucleotide NMN,the intermediate of nicotinic acid and nicotinamide metabolism pathway for the follow-up basic research.3.It is verified at the cellular level that NMN can activate AMPK signaling pathway,inhibit m TOR/p70S6 K pathway,activate autophagy and promote iron death,thus inhibiting the proliferation and promoting the apoptosis of liver cancer cells.4.The animal level verified that nicotinamide mononucleotide(NMN)depressed the growth of hepatoma cell line transplanted tumor model in nude mice by increasing the level of NAD+ in tumor tissue,and promoted the apoptosis of hepatoma cells.