Lactobacillus casei relieves liver injury by regulating immunity and suppression of the enterogenic endotoxin‐induced inflammatory response in rats cotreated with alcohol and iron

Abstract Excessive alcohol and iron intake can reportedly cause liver damage. In the present study, we investigated the effect of Lactobacillus casei on liver injury in rats co‐exposed to alcohol and iron and evaluated its possible mechanism. Sixty male Wistar rats were randomly divided into three groups for 12 weeks: the Control group (administered normal saline by gavage and provided a normal diet); alcohol +iron group (Model group, treated with alcohol [3.5–5.3 g/kg/day] by gavage and dietary iron [1,500 mg/kg]); Model group supplemented with L. casei (8 × 108 CFU kg−1 day−1) (L. casei group). Using hematoxylin and eosin (HE) staining and transmission electron microscopy, we observed that L. casei supplementation could alleviate disorders associated with lipid metabolism, inflammation, and intestinal mucosal barrier injury. Moreover, levels of serum alanine aminotransferase, gamma‐glutamyl transferase, triglyceride (TG), and hepatic TG were significantly increased in the model group; however, these levels were significantly decreased following the 12‐week L. casei supplementation. In addition, we observed notable improvements in intestinal mucosal barrier function and alterations in T lymphocyte subsets and natural killer cells in L. casei‐treated rats when compared with the model group. Furthermore, L. casei intervention alleviated serum levels of tumor necrosis factor‐α and interleukin‐1β, accompanied by decreased serum endotoxin levels and downregulated expression of toll‐like receptor 4 and its related molecules MyD88, nuclear factor kappa‐B p65, and TNF‐α. Accordingly, supplementation with L. casei could effectively improve liver injury induced by the synergistic interaction between alcohol and iron. The underlying mechanism for this improvement may be related to immune regulation and inhibition of enterogenic endotoxin‐mediated inflammation.


| INTRODUC TI ON
The liver is an essential organ for alcohol metabolism and iron storage; therefore, it is the primary target organ for alcohol injury and iron overload (Lainé et al., 2017;Lakhal-Littleton et al., 2016). The intake of both excessive alcohol and iron (iron or iron-rich foods) can lead to liver damage via oxidative stress and lipid peroxidation (Osna et al., 2017;Pietrangelo, 2016); excessive alcohol intake can also promote iron absorption, resulting in excessive iron deposition in the liver (Corradini & Pietrangelo, 2012;Grochowski et al., 2019). In addition, studies have revealed that co-exposure to alcohol and iron results in synergistic oxidation and cumulative effects that aggravate hepatocyte injury (Fletcher & Powell, 2003;Sumida et al., 2001).
Reportedly, the immune status of T lymphocytes is closely related to the occurrence and development of alcoholic liver injury (Matos et al., 2013). Long-term drinking can inhibit innate immune cells and reduce the number and activity of lymphocyte subsets (Støy et al., 2015). Alcohol consumption also impacts the functions of hepatic natural killer (NK) cells (Cui et al., 2017). In addition, disturbances in iron homeostasis have been associated with altered immune functions and liver injury. Iron overload can affect lymphocyte proliferation/maturation, induce the apoptosis of T lymphocytes, and selectively affect peripheral T lymphocytes, with hepatocellular ballooning injury (Buracco et al., 2017;Handa et al., 2016). Based on these previous reports, it can be postulated that the immune state caused by excessive alcohol and iron intake mediates the development of liver injury.
Endotoxins are a major component of Gram-negative bacterial cell walls (Clementi et al., 2017). Notably, excessive alcohol intake is known to result in enhanced intestinal mucosal permeability, which, in turn, causes the leakage of large amounts of gut-derived endotoxins from the gut lumen into the systemic circulation (Meroni & Longo, 2019). These endotoxins enter the liver through the portal vein and bind to toll-like receptor 4 (TLR4) on the Kupffer cell surface. Subsequently, a cascade reaction triggers downstream signaling molecules, including MyD88 and nuclear factor kappa-B (NF-κB), resulting in the activation of Kupffer cells to secrete and release excessive proinflammatory cytokines such as tumor necrosis factor (TNF)α and interleukin (IL)-1β (Li et al., 2012;Zannetti et al., 2016), which ultimately causes inflammatory injury to hepatocytes.
Additionally, excessive iron uptake by macrophages can enhance NF-κB activity, further promoting inflammatory factor production and intensifying liver injury (Liu Meng-na & Zhang, 2019).
Probiotics are living microorganisms that are beneficial to the health of the host. Appropriate probiotic supplementation improves the repair of intestinal mucosal damage (Deng et al., 2017) and affords anti-inflammatory (Li et al., 2016), antioxidant (Wang et al., 2017), and immune regulation (La Fata et al., 2018). As a well-known probiotic, Lactobacillus casei, in addition to the above characteristics, has attracted extensive attention, especially for its protective effect against alcoholic liver injury. Several studies have achieved good therapeutic effects following L. casei supplementation in patients with alcoholic cirrhosis (Koga et al., 2013;Macnaughtan et al., 2020;Stadlbauer et al., 2008), during which iron deposition is a known marker (Zhang & Krinsky, 2004). Currently, the mechanism underlying the protective effect of L. casei on alcoholic liver injury remains poorly understood. Accordingly, we aimed to explore the effects of L. casei on liver injury in rats induced by the synergistic interaction between alcohol and iron. Furthermore, we investigated the mechanism underlying immune regulation and inhibition of enterogenic endotoxin-mediated inflammation.

| Animals and ethics statement
Adult male Wistar rats (180-220 g, aged 2 months) were provided by the Animal Experiment Centre (Qingdao, China). The experimental protocol was approved by the Animal Ethics Committee of Qingdao University.

| Experimental design
After one week of adaptive feeding, 60 male Wistar rats were randomly divided into 3 groups (20 animals/group): the control group, normal saline by oral gavage and a standard diet; the Model group (alcohol +iron group), fed a standard diet containing high dietary iron (1,500 mg/kg) and 56% v/v alcohol by oral gavage (3.5g kg −1 day −1 2 weeks +5.3g kg −1 day −1 10 weeks); the L. casei group (L. casei treatment group), fed a standard diet containing high dietary iron (1,500 mg/kg) and oral alcohol and L. casei (the dose of alcohol was the same as in the model group; L. casei, 8 × 10 8 CFU/kg −1 day −1 ). The experiment was performed over a 12-week period.
After 12 weeks, the animals were sacrificed, and the blood (serum or plasma), liver, and small intestinal tissues were harvested and stored at −80°C. Fresh liver tissues were quickly excised, fixed in 10% formaldehyde, and embedded in paraffin.

| Determination of serum iron (SI), liver iron concentration (LIC), inflammatory factors, endotoxin, liver function, and lipid metabolism
The levels of SI and LIC were measured using inductively coupled plasma mass spectrometry (ICP-MS) by strictly following the manufacturer's instructions. In addition, the levels of interleukin-1β (IL-1β), tumor necrosis factorα (TNFα), and endotoxin were measured by a competitive enzyme immunoassay using an enzyme-linked immunosorbent assay (ELISA) kit (Cloud-Clone Corp, Katy, USA), in accordance with the manufacturer's instructions.

| Histopathological analysis
The formalin-fixed liver tissues were subjected to hematoxylin-eosin (HE) staining, and morphological changes were examined under a light microscope (Olympus BX60, Tokyo, Japan). Ultrastructures of the small intestinal and liver tissues were observed using a JEM-1200EX transmission electron microscope (TEM; JEOL, Tokyo, Japan).

| Detection of T lymphocyte subsets and NK cells
The number of CD4 + , CD8 + T lymphocyte subsets, and NK cells in the peripheral blood of rats was determined flow cytometrically. Monoclonal antibodies labeled with fluorochrome, TCRαβ-FITC, CD3-FITC, CD4-PE, and CD8-PE were procured from BD Pharmingen. For each test, 100 μl of fresh heparinized rat whole blood was incubated with indicated antibodies for 15 min, then lysed with FACS™ lysing solution, washed with phosphate-buffered saline, fixed, and eventually detected by BD FACSAria with DB FACSDiva software.

| Small intestine tracer permeability assay
In brief, the ends small intestinal middle segment tissues (2 cm) were ligated, and 2 mg/ml EZ-link Sulfo-NHS-Biotin was slowly injected; this was followed by incubation for 5 min at room temperature. The tissues were fixed in 4% paraformaldehyde. After 24 hr, the sections were washed three times with phosphate-buffered saline, embedded in paraffin, and cut into 5μm thick sections. The sections were incubated with streptavidin DyLightTM 488 conjugated (1:500 dilution) for 30 min in the dark. The distribution of EZ-link Sulfo-NHSbiotin was observed using a fluorescence microscope.

| Expressions of TLR4 and MyD88 signaling pathway-related proteins by Western blot analysis
Proteins were extracted from liver tissues using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Jiangsu, China), in accordance with the manufacturer's instructions. Total protein content was determined using the BCA Protein Assay Kit (Beyotime).
Subsequently, membranes were washed three times with TBST for 10 min, followed by incubation with the corresponding secondary antibodies (1:3,000 dilution; Zhongshan Goldenbridge, Beijing, China) for 2 hr at 37°C. Finally, protein bands were visualized using an enhanced chemiluminescence detection kit (Beyotime). Histone H3 and β-actin served as internal controls for nucleoprotein and cytoplasmic proteins, respectively.

| Statistical analysis
All data values are expressed as mean ±standard deviation using SPSS (version 18; IBM Corp., Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to compare multiple groups, followed by Duncan's multiple range test. p ˂ 0.05 was deemed statistically significant.

| Effects of L. casei on body weight
The recorded body weights before and after the intervention are shown in Figure 1. No significant difference in body weight was observed between the groups before intervention (p > .05). However, the weight of the model group reached (348.17 ± 29.18) g after the 12-week intervention, which was significantly decreased when F I G U R E 1 Effects of L. casei on body weight. Control: the control group; Model: the model group; Lactobacillus casei: the L. casei group. * p < .05 versus the control group, # p < .05 versus the model group compared with the control group (401.17 ± 18.93) g (p < .05).
Lactobacillus casei supplementation significantly improved the weight loss observed in rats cotreated with alcohol and iron (p <.05) ( Figure 1).

| Effects of L. casei on SI and LIC
As shown in Figure 2, SI and LIC were significantly increased (58.17% and 24.46%, respectively) in the model group when compared with those in the control group (p < .05); these levels were significantly decreased following L. casei supplementation by 20.60% and 24.65%, respectively (p < .05).

| Effects of L. casei on pathological changes in the liver
We examined liver sections from each group using HE staining and light microscopy. The control group presented a typical lobular structure, with an ordered hepatic cord, normal hepatocyte structure, and the absence of steatosis or inflammatory infiltration The control group displayed a normal mitochondrial structure, with clearly visible cristae, regularly arranged endoplasmic reticulum, and typically shaped bile canaliculi (Figure 4a). In the model group, large amounts of lipid droplets were observed. The bile canaliculi and microvilli were swollen, and the mitochondrial ridge was slightly blurred. In addition, the endoplasmic reticulum was arranged in a disorderly fashion (Figure 4b). Supplementation with L. casei alleviated the swelling in the microvilli and bile canaliculi.
Structurally, mitochondria and endoplasmic reticulum tended to be normal, with only a few lipid droplets observed (Figure 4c). This indicated that L. casei supplementation could alleviate the abnormal liver tissue ultrastructure induced by cotreatment with alcohol and iron in rats.

| Effects of L. casei on liver function and lipid metabolism
As shown in Figure 5, the serum levels of ALT, GGT, TG, and hepatic TG were increased by 116.7%, 50.42%, 78.97%, and 86.08%, respectively, in the model group when compared with those in the control group (p < .05); these levels were significantly decreased by 33.33%, 38.59%, 32.38%, and 28.31%, respectively, (p < .05) after Lactobacillus casei supplementation.

| Effects of L. casei on T lymphocyte subsets and NK cells
We observed that the CD4 + lymphocyte percentage and the CD4 + / CD8 + ratio in the model group were significantly decreased when compared with the control group, while the CD8 + lymphocyte percentage and NK cell lymphocyte percentage were significantly increased (p < .05). Lactobacillus casei supplementation significantly decreased the percentage of CD8 + lymphocytes and NK cells (p < .05); simultaneously, the CD4 + /CD8 + ratio was significantly increased (p < .05). Accordingly, L. casei supplementation could effectively improve the immune response in rats cotreated with alcohol and iron ( Figure 6, Table 1).

| Effects of L. casei on endotoxin
As shown in Figure 9, the serum level of endotoxin in the model group was significantly increased by 72.41% when compared with that in the control group (p < .05); however, this level was significantly decreased by 22% (p < .05) following L. casei supplementation.

| Effects of L. casei on TLR4 signaling pathway and inflammation
As shown in Figure 10, the expression of TLR4, MyD88, TNFα, and NF-κB p65 proteins was upregulated following cotreatment with alcohol and iron when compared with the control group (p < .05).
The serum levels of TNFα and IL-1β in the model group were significantly increased by 25.28% and 27.36% when compared with those in the control group (p < .05); these levels were significantly decreased by 14.36% and 30.17%, respectively (p < .05), following L. casei supplementation.

| D ISCUSS I ON
Epidemiological studies have indicated that excessive intake of alcohol and iron can damage liver functions. Alcohol is a leading cause of liver diseases worldwide (Leggio & Lee, 2017). The number of proliferating hepatocytes was significantly increased in mice fed an excess-iron diet, with the iron overload reportedly inducing mitochondrial injury (Furutani et al., 2006). Even under low alcohol intake, a certain amount of iron overload can cause significant liver damage (Gao et al., 2017). In the present study, a rat model of liver injury was established by co-administering Herein, we observed that L. casei significantly improved the weight of rats treated with alcohol plus iron, indicating that nutritional intake and absorption in rats were affected. This finding is consistent with our previous report (Ma et al., 2018). One possible explanation is damage to liver function. In addition, serological and pathological examinations indicated that the liver damage caused by combined exposure to alcohol and iron, including liver function decline, lipid metabolism disorders, and inflammatory cell infiltration, were significantly alleviated by L. casei supplementation, with a significant reduction in SI and MIC. These results indicate that L. casei could effectively improve liver injury in rats induced by the synergistic interaction between alcohol and iron.
Previous studies have revealed that liver injury is closely asso-  (Kawabata et al., 2008;Qu et al., 2015). In addition, studies have shown that iron overload is closely related to  (Song et al., 2018). We have previously reported that L. casei can regulate the proportion of T lymphocyte subsets and NK cells, thus improving the immune function of rats with alcoholic liver injury or breast cancer (Zhengyan et al., 2017;Yiyun et al., 2019). The findings of our present study indicate that L. casei supplementation significantly decreased the percentage of CD8 + lymphocytes and NK cells, while the CD4 + /CD8 + ratio was significantly increased. This suggests that the protective effect of L. casei on alcohol plus iron-induced liver injury may be related to the proportion of CD4 + and CD8 + T lymphocyte subsets and NK cells regulated by L. casei.
Previous studies have revealed that enterogenic endotoxinmediated inflammation is involved in the progression of alcoholic liver injury. Xiao et al. have demonstrated that treatment with rice bran phenolic extract represses the alcohol-induced trigger of the hepatic endotoxin-TLR4-NF-κB pathway, followed by mitigated liver inflammation (Xiao & Zhang, 2020 (LPS)-induced liver injury and inflammation (Perea et al., 2017).
Moreover, iron overload-induced inflammation causes further damage to the liver tissue (Preziosi et al., 2017). Our previous studies have revealed that L. casei improves intestinal injury induced by acrylamide in rats and D-galactose in aging mice (JiaH Fang et al., 2016;Tianjiao et al., 2018). In the present study, TEM and tracer experiments showed that L. casei supplementation could effectively repair the intestinal mucosal barrier damage induced by co-exposure to alcohol and iron in rats. We observed that the in- The TLR4 MyD88 signaling pathway plays a vital role in the inflammatory response (Ikebe et al., 2009;Takeda et al., 2003). Based on the above evidence, we speculate that the inhibition of inflammation by suppressing the enterogenic endotoxin-mediated TLR4 signaling pathway could be one of the possible protective mechanisms of L. casei on liver injury in rats induced by the synergistic interaction between alcohol and iron.

| CON CLUS IONS
In summary, our findings demonstrate that L. casei supplementation could effectively improve liver injury induced by the synergistic interaction between alcohol and iron. The underlying mechanism may involve improved immunity and inhibition of enterogenic endotoxinmediated inflammation. These findings provide a scientific basis for developing novel treatment strategies for alcoholic liver disease with coexisting iron overload.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.