In the present study, we preliminarily explored the effect of L-P-cs on CCl4-induced ALI in mice. We found that L-P-cs synergistically inhibited liver lobule necrosis, hepatocyte inflammation, and apoptosis by reducing liver oxidative stress and ERS.
Recent studies found that probiotics have a therapeutic effect on alcoholic liver disease, but their unstable nature and toxic side-effects have greatly limited their clinical application[31]. Studies confirmed that biologically-active metabolites produced by probiotics such as short-chain fatty acids, extracellular polysaccharides, and neuroactive metabolites have antioxidant, anti-inflammatory anti-tumor properties that benefit the host[32]. Probiotics also produce a variety of effective bacteriocins that may be used as food preservatives or antibiotic supplements[17]. These lines of evidence suggest that probiotic metabolites have the potential to serve as hepatoprotective microorganisms and may be less affected by environmental factors and more stable.
Lactobacillus plantarum ST-III is a probiotic with cholesterol-lowering activity[18]. Therefore, we hypothesized that Lactobacillus plantarum ST-III culture supernatant (L-P-cs) might have unexpected liver-protective effects. Our findings supported this hypothesis. Pretreatment with L-P-cs prevented alterations in serum levels of transaminases and HMGB1 in mice with acute CCl4-induced liver injury. Histology showed that CCl4 induced ALI in mice characterized by liver lobule congestion, inflammatory cell infiltration, and increased mitotic activity. These effects were significantly prevented by L-P-cs pretreatment. These are only a subset of some pathological results; therefore, we further explored the liver-protective mechanisms of L-P-cs.
After CCl4 enters the body, it is metabolized to produce chloroform free radical (CCl3·), which causes lipid peroxidation in liver cells and damage to cell membranes and organelles under the influence of liver cytochrome P450. Recent research in free radical biology suggested an essential pathophysiological role of free radicals and oxidative stress in developing and progressing liver diseases[33]. Classical CCl4-induced ALI in mice can be divided into two stages: the first stage is liver oxidative and lipid peroxidation damage. This damage leads to deficiency or depletion of endogenous antioxidant enzymes; therefore, liver oxidative injury can be assessed by measuring levels of the lipid peroxidation product MDA, superoxide dismutase (SOD), and GSH in liver tissue. MDA is an essential product of oxidative stress[34]. We found that L-P-cs significantly increased GSH-Px, T-AOC, and SOD in liver tissue, while MDA content decreased. These changes suggest that L-P-cs prevents liver injury by reducing oxidative stress.
Several studies confirmed that Nrf2 is highly expressed in the liver. When ROS or electrophiles attack cells, Nrf2 dissociates from Keap1 and translocates into the nucleus, first forming a heterogeneous relationship with the small Maf protein[35, 36]. The dimer, combined with the antioxidant response element, activates transcriptional activation of the antioxidant enzyme gene expression regulated by Nrf2. We found that CCl4 stimulation reduced liver Nrf2 expression, and L-P-cs pretreatment significantly normalized these changes. We performed western blot analysis of the expression of HO-1, NQO1, and SOD2 downstream of Nrf2 and found that L-P-cs' antioxidant effect was achieved via the Nrf2 signaling pathway.
The second stage is CCl4-induced inflammatory liver injury. Free radical metabolism not only directly damages liver tissue but also promotes inflammation. Therefore, CCl4-induced liver inflammatory injury can be assessed by measuring the expression of the inflammatory factors TNF-α and IL-6 in serum and liver tissue. We found that the expression of TNF-α and IL-6 increased significantly in the injury group but decreased in the L-P-cs treatment group, suggesting that L-P-cs prevents CCl4-induced liver injury by reducing inflammation.
ERS inhibits fatty acid oxidation in the liver[37]. We found that CCl4-induced ALI also caused ERS and hepatocyte calcium overload. Intracellular calcium overload has been shown to induce cell death through ERS[38]. The mitochondria absorb the Ca2+ released by the ER, and then the permeability transition pore is opened to release cytochrome c and activate the apoptotic caspase cascade[39]. At the cellular level, Lactobacillus plantarum ST-III promotes intracellular Ca2+ assimilation[40]. Therefore, we hypothesized that L-P-cs' protective effect on the liver would also be mediated by promoting intracellular Ca2+ assimilation to reduce ERS. This result was verified by our experiment involving the ATF6 signaling pathway.
Activated ATF6 enters the nucleus and binds to the promoter ER stress response element as a homologous or heterodimer with the universal transcription factor nuclear factor-Y. This binding induces the transcriptional expression of ERS genes such as GRP78, PDI, and CHOP[41] to protect cells from damage caused by ER and restore cell function[42].
GRP78 is an endoplasmic reticulum chaperone protein. GRP78 is an essential protein to main cellular homeostasis via ca-ATPase. Since CCl4 can directly act on mitochondria, the GRP78 increase might result from stress response that occurred in the early stage[43]. Abnormal expression of GRP78 in the endoplasmic reticulum leads to misfolding. We found that CCl4 treatment elevated levels of ERS markers ATF6, GRP78, PDI, and CHOP; after pretreatment with L-P-cs, these protein levels tended to normalize. These findings suggest L-P-cs resists CCl4-induced ALI by regulating ERS in liver cells.
Ca2+ released by ER activates the caspase cascade of apoptosis that induces hepatocyte death. Early studies showed that oxidative stress is involved in apoptosis and can be triggered by promoting ROS production and reducing antioxidant function[44]. In this context, we explored the effect of CCl4-induced ALI on apoptosis. The pro-apoptotic protein Bax is overexpressed in CCl4-induced ALI, and expression of its related anti-apoptotic protein Bcl-2 is suppressed, suggesting that CCl4 exposure causes severe cell death. Importantly, through L-P-cs preprocessing, this situation is significantly improved. The ability of L-P-cs to reduce apoptosis was verified again in terms of Cleaved-caspase3. This finding also suggests that L-P-cs inhibits ERS through intracellular Ca2+ assimilation, thereby reducing cell apoptosis.
Caspases comprise a large family. We only explored Cleaved-caspase 3 under L-P-cs pretreatment, and this is insufficient. Further studies will determine whether L-P-cs pretreatment reduces apoptosis by affecting other factors levels in liver cells. Apoptosis is programmed cell death characterized by specific morphological features, including cell shrinkage, nuclear fragmentation, and chromatin condensation[43]. Indeed, this study confirmed that Cleaved-caspase 3 and Bax levels were increased in CCl4-induced mice in vivo, as well as in NCTC1469 cells in vitro. Levels of Bcl-2 expression were lower in CCl4-treated mice in vivo and NCTC1469 cells in vitro. Together, in vivo and in vitro results suggested that L-P-cs reversed these apoptotic indicators; this tendency ameliorated CCl4-induced liver apoptosis and contributed to its hepatoprotective activity.
In summary, our findings suggest that L-P-cs protect against CCl4-induced ALI. This finding was demonstrated by suppressing oxidative stress and ERS to reduce hepatocyte inflammation and apoptosis. It is generally believed that the intake of large amounts of probiotics entails many unstable factors that are reflected in the instability of its biological characteristics and their possible unpredictable side effects[31, 45, 46]. Although our research is currently limited to an overview of L-P-cs through the cell signal transduction system to treat CCl4-induced ALI, it provides novel insights regarding L-P-derived products that reduce the development risk of CCl4-induced ALI. Future studies focusing on probiotic supernatants may help broaden the scope of clinical use of probiotics (Fig. 7)