This study has revealed that there may be an interaction of gut microbiota, related metabolites and inflammation factors with NAFLD. In addition, this study assessed the efficacy of probiotic therapies in modifying inflammation factors in NAFLD.
As regard to gut microbiota and gut metabolites, although gut microbiota of humans was highly variable, this review has shown that patients with NAFLD reported more harmful bacteria (such as Gammaproteobacteria) and less beneficial bacteria (such as Bifidobacteriaceae) than the control group17. Considering several contradictory findings of related studies with similar designs (such as Lachnospiraceae and Prevotellaceae), additional large-scale studies on the gut bioactive metabolites and metagenomics characteristics of gut microbiota in NAFLD are needed to confirm these results16.
It has long been known that high-fat diet could induce gut dysbiosis, and prolonged gut dysbiosis could produce different gut metabolites, including trimethylamine, SCFAs, glucagon-like peptide 1, lipopolysaccharide, free fatty acids, free cholesterol, and ethanol10. In addition, these gut metabolites may promote systemic inflammatory response, such as lipopolysaccharide, which can activate Kupffer cells to produce TNF-α and cause insulin resistance13. Thus, insulin resistance increases the accumulation of hepatic triglycerides by promoting triglyceride synthesis, hepatic uptake of free fatty acids and peripheral lipolysis in NAFLD7. Therefore, patients affected by NAFLD may have a gut dysbiosis, accompanied by small intestine bacterial overgrowth and elevation of gut metabolites such as endotoxin. Noteworthy, increased intestinal permeability may lead to increased bacterial migration and, under this condition, gut metabolites and some bacteria may enter the portal circulation by permeating the intestinal barrier and reach the liver12,18. Clinical studies have also shown that almost all patients with NAFLD had abnormal levels of inflammatory cytokines, which triggered the inflammatory response pathway of the gut microflora in NAFLD. Furthermore, experimental studies have shown that cytokine-mediated oxidative stress, mitochondria and endoplasmic reticulum dysfunction may promote the development of steatohepatitis from simple steatosis via the toll-like receptor signaling pathway12,19.
Accumulated evidence suggested that oxidative stress played an important role in the multi-hit hypothesis model of progression of NAFLD18. At the organelles level, mitochondria and endoplasmic reticulum are the main sites of ROS formation, producing pro-apoptotic peroxidized lipids (4-hydroxy-2-nonenal) and Bax, and promoting the synthesis of respiratory proteins through PGC1α as well as the transfer of Ca2+ and ONOO20,21. Consequently, these may promote activation of stellate cells, macrophages, and Kupffer cells, which further promote endoplasmic reticulum and mitochondrial dysfunction, hence inducing inflammation, fibrosis and apoptosis in NAFLD12. Therefore, these results supported the hypothesis that interactions among the liver, the immune system and the gut microflora related metabolites have a key role in the development of NASH from simple steatosis. An illustration of this hypothesis is shown in Fig. 4 (by KA).
Considering the gut metabolites and inflammatory factors in NAFLD, accumulating evidence has found that gut microbiota communities generate a variety of metabolites, which are bioactive substances that interact with the host liver cells in NAFLD13. Among them, endotoxin and lipopolysaccharide are active metabolites of gram-negative bacterial envelope components, and significantly increase the (mainly through activation of TLR4 signaling pathway) release of a range of inflammatory factors, including IL-6, IL-12, IL-1β and TNF-α in the development of NAFLD22. Additionally, exposure of the liver to endotoxin and lipopolysaccharide causes pathological and metabolic changes that induce an acute inflammatory response and early accumulation of inflammatory cells, such as monocytes, neutrophils and lymphocytes. Moreover, inflammatory cells release proteases, other enzymes and reactive oxygen metabolites, leading to pathological progression of liver injury in NAFLD.
Furthermore, bile acids are regulators of lipid, glucose metabolism, and modulate inflammation in NAFLD. Therefore, the gut microbiota converts the primary bile acids (including chenodeoxycholic acid and cholic acid at the colon and distal small intestine) to secondary bile acids, such as ursodeoxycholic acidursodeoxycholic acid, lithocholic acid, and deoxycholic acid23. Also, bile acids can regulate the composition of gut microbiota, which in turn can regulate the capacity and composition of bile acids24. Thus, the destruction of bile acid-flora interaction can promote the development of NAFLD inflammation25. Meanwhile, studies have shown that farnesoid X receptor (FXR) plays an important role in bile acid-flora interactions. Therefore, bile acids are the ligands for the FXR, a member of a family of nuclear hormone receptors, widely found in the ileum and liver that regulate a variety of metabolic pathways26. The regulation of FXR signal has become a potential target for the prevention and treatment of NAFLD and related metabolic disorders26. Furthermore, not only do bile acids and cholic acids have direct antibacterial properties, but also mitigate inflammatory response through the inhibition of NF-κB signaling and cytokines generation in macrophages, mainly by activating Takeda-G-protein-receptor-527. Meanwhile, peptidoglycan from gram-positive bacteria promotes inflammatory response, primarily through the activation of mitogen-activated protein kinase (MAPK)/NF-κB of the mediated proinflammatory cytokine generation. Subsequently, it also plays an important role in activating immune cells such as NK cells, dendritic cells, macrophages, B cells and macrophages. In addition, bacterial DNA activates MAPK/NF-κB (mainly through activation of TLR9 signaling pathway)28.
It is noteworthy that the hepatic steatosis is considered to be the benign beginning of the NAFLD, which is reversible without serious liver injury at this stage. Therefore, the preceding discussion in relation to the results of this study clarifies the roles of gut microbiota, related metabolites and inflammation factors in the pathophysiological process of pure steatosis of liver to NASH. It is hoped that this evidence may provide new insights for future researches and development of drugs targeted at preventing the hepatic steatosis progression to NASH, steatohepatitis-related associated cirrhosis and hepatocellular carcinoma. Interestingly, a recent meta-analysis also showed that probiotics reduced liver transaminase, total cholesterol, TNF-α, and insulin resistance in NAFLD patients29. However, that meta-analysis analyzed studies which considered only one inflammatory factor, TNF-α. In this regard, that meta-analysis did not investigate other inflammatory factor levels with respect to the association between probiotics and NAFLD, which has been addressed in this study.
Therefore, this study has shown that there is an indirect evidence of the effects of the probiotic (beneficial gut microbiota) interventions on inflammation and liver damage, which indicated significant reductions in inflammatory factors (CRP and TNF-α) in NAFLD after probiotics were used. Nevertheless, there was no statistically significant difference in the concentrations of IL-6 between the NAFLD patients and the controls, which may be attributed to the small sample sizes involving IL-6. Therefore, large-scale clinical studies on this phenomenon are needed in the future.
Additionally, a systematic review suggested that pentoxifylline, an anti-TNF-α agent, directly targets the inflammatory process. In this regard, reduced AST and ALT levels may improve liver histological scores in patients with NALFD/NASH30. However, anti-TNF-α therapy for NAFLD may have significant side effects. Therefore, large, prospective and well-designed randomized controlled studies are needed to explore medications with better efficacy for NAFLD. Besides, new therapeutic targets for inflammatory signaling pathways are also worth exploring, as well as the treatment of NAFLD with CRP as the target.
Also, in agreement with the findings of this study, some studies have shown that activated carbon could reduce metabolites in NAFLD. For example, in addition to the probiotic supplementation, Yaq-001 (Yaqrit Ltd.) carefully designed and developed a novel synthetic (both macroporous and microporous) activated carbon for treating gut microbiota and related metabolites modulation in NAFLD31. In this regard, it has been shown that Yaq-001 (when administered orally) could reduce the transintestinal migration of gut microbiota and related metabolites, such as dimethylarginine, ammonia, bacteria-derived products, acetaldehyde, hydrophobic bile acids, and inflammation factors, including TNF-α and IL-631. A safety and efficacy study on Yaq-001 is being conducted through the European Commission Horizon program. A clinical trial is also under way (NCT03962608). Future studies should focus on developing more specific novel therapeutics for NAFLD based on bioengineering technology with high efficacy and specificity. For example, utilizing targeted engineered individual microbiota instead of fecal transplants, or utilizing engineered microbiota capable of producing anti-inflammatory or antioxidants molecules to replace the antibiotics, or utilizing engineered compounds that modulate or adsorb the gut microbiota-derived metabolites of interest.
There are some limitations of this study, which should be acknowledged. Firstly, the heterogeneity was very high in the meta-analysis of TNF-α. Given that the subjects in this study came from different countries and ethnic groups, this heterogeneity may be due to the large individual differences in TNF-α concentrations, especially considering the significant side effects of TNF-α in the pathogenesis of NAFLD, hence the findings on TNF-α in this study should be interpreted with caution. Secondly, this study only analyzed the overall level of each study and did not quantify the important clinical indicators of individual patient level (also due to the lack of information in the original study), such as race, smoking, alcohol drinking and blood pressure, which could also be a reason for the large heterogeneity. Thirdly, the probiotic intervention was a well-designed experiment, and whether it is possible to replicate the results in the general population is unknown. Therefore, large population cohort studies are needed in the future to provide a causal relationship between intestinal flora and its metabolites and NAFLD.
Although there is currently no efficient treatment available for NAFLD, the findings of this systematic review/meta-analysis suggest that the gut microbiota, related metabolites and inflammation factors (especially CRP and TNF-α) network presently appear to be innovative and potentially promising treatment targets. Future studies should focus on developing more specific novel therapeutics for NAFLD based on bioengineering technology with high efficacy and specificity.