Discussion
Our study showed that NAFLD patients with elevated intestinal permeability have more severe disease status, manifested as more serious liver dysfunction, hyperlipidemia, liver fat deposition, insulin resistance, and intestinal barrier damage. Our data also showed that serum D-lactate is positively correlated with parameters indicative of disease severity, including ALT, AST, GGT, TBIL, IBIL, FAP, and TG. These findings reveal a clear association between intestinal permeability and disease severity in patients with NAFLD, which add to accumulating evidence supporting the “gut-liver axis.” Hence, intestinal manifestations warrant increased attention in patients with severe NAFLD such as NASH and NAFLD-associated cirrhosis.
However, a previous study with a small sample size (35 NAFLD patients and 24 controls) reported no significant differences in liver transaminases and TG between NAFLD patients with normal and increased intestinal permeability, measured by 51Cr-EDTA excretion testing [15]; while another study reported a correlation between intestinal permeability and pathologic severity (portal inflammation, fibrosis, and ballooning of hepatocytes) using the lactulose-mannitol bowel permeability test, in line with our findings [12]. Further, zonulin, a moderator of intestinal permeability, was found to be positively correlated with some parameters of disease severity, such as ALT, TG, HOMA-IR, and liver histopathology in patients with NAFLD, hence indicating a correlation between intestinal permeability and NAFLD severity [25]. In addition to the small sample size of the previous study being likely inadequate to show the relationship between intestinal permeability and disease severity in patients with NAFLD, using different methods to detect intestinal permeability may also lead to inconsistent results. In addition to51Cr-EDTA excretion testing and lactulose-mannitol bowel permeability test, testing for D-lactate levels has become a widely used method for intestinal permeability detection in recent years [23, 24]. Owing to the simplicity of this method, the bias caused by multiple operations can be reduced. However, given that the criteria for defining intestinal permeability have not reached certain consensus, other tests to evaluate intestinal permeability should be used to confirm these findings by further studies.
There are two plausible reasons for the observed association between intestinal permeability and severity of NAFLD. First, elevated intestinal permeability can cause pathogenesis and progression of NAFLD. Animal research has shown that increased intestinal permeability induced by dextran sulfate sodium (DSS) enhances high-fat diet-induced hepatic inflammation and steatosis in mice [26]. Subsequent to the increase of intestinal permeability, bacterial components, particularly LPS, can translocate into the portal vein and thus the liver, resulting in liver inflammation and injury [27, 28]. In support of this hypothesis, a clinical study showed that plasma antibodies against LPS were increased in patients with NASH compared with healthy controls, and increased with aggravated inflammation in NASH, indicating an association between LPS exposure and the severity of NASH in humans [29]. Consistent with this finding, our data showed a positive correlation between D-lactate and LPS, although the difference did not reach the threshold for statistical significance (P=0.069), suggesting that increased intestinal permeability may lead to LPS translocation, and consequently enhanced liver injury. The alternative explanation is that NAFLD may contribute to the increase in intestinal permeability, as disruption of the intestinal epithelial barrier and gut vascular barrier can be detected in NAFLD mice induced by high-fat diet [14].
Recent studies have found that improvement of intestinal permeability by fecal microbiota transplantation or probiotics had a therapeutic effect on NAFLD [16, 17]. These results suggest that improvement of intestinal permeability may promote the lipid-lowering effect of metabolic therapy in patients with NAFLD. Accordingly, our data revealed that NAFLD patients with elevated intestinal permeability present with less substantial improvement in TG levels after metabolic therapy. This study has provided evidence for the role of elevated intestinal permeability in NAFLD progression and suggested that a combination of treatment to improve intestinal barrier and metabolic therapy may have better therapeutic effects on NAFLD patients, especially those with elevated intestinal permeability.
However, we found that the improvement of clinical characteristics, liver test parameters, and liver ultrasonographic parameters were not significantly different between NAFLD patients with elevated and normal intestinal permeability after one month of metabolic treatment. As these patients received metabolic therapy, the improvement of blood lipids was more obvious, while other parameters of disease severity did not show significant improvement. In addition, we only analyzed the results of the one-month-long metabolic therapy, thus the unexceptional improvement in other parameters could likely be attributed to the short treatment time. Therefore, a prospective and long-term follow-up study is needed to assess whether intestinal permeability affects the improvement of other parameters in patients with NAFLD after treatment.
The present study has some limitations. First, intestinal biopsies were not performed as standard in our investigation, as most enrolled patients had no gastrointestinal symptoms. Second, because this is a retrospective study, some potential confounders (e.g., use of medication with liver toxicity) that may lead to liver injury and steatosis were not recorded. Third, although our sample size was larger than those of previous studies investigating the association between intestinal permeability and NAFLD, this was a single-center study with a limited sample size. Only 30 patients’ clinical data were available for analyzing the efficacy of metabolic therapy in NAFLD patients with normal or elevated intestinal permeability; moreover, we could not analyze whether intestinal permeability affects the improvement of other parameters in patients with NAFLD after treatment. Therefore, our results should be interpreted with caution, and future studies are needed to confirm these findings.
In summary, intestinal permeability correlates with the severity of liver dysfunction, hyperlipidemia, liver fat deposition, insulin resistance, and intestinal barrier damage in patients with NAFLD. Moreover, intestinal permeability may be valuable for predicting the efficacy of metabolic therapy in patients with NAFLD.
 Acknowledgement
The manuscript has been presented as a preprint.