Preventive Effect of Lactobacillus Plantarum HFY15 on Oxidative Damage and Acute Liver Injury Induced by Carbon Tetrachloride (CCl 4 ) in Mice

Background and bulgaricus (LDSB) for two weeks, and intraperitoneal injection of CCl 4 -induced acute liver injury to study the preventive effect of LP-HFY on CCl 4 -induced acute liver injury, especially in oxidative damage.

the host [6]. Lactic acid bacteria is a microbial species widely used as probiotics in the food industry. It has been proven to have multiple functions such as regulating skin health [7], reducing memory impairment in mice [8], and inhibiting enterovirus Coxsackie virus B4 (CV-B4) [9], Other studies have shown that Lactobacillus fermentum LA12 can be used to prevent and treat alcoholic liver injury [10]; Lactobacillus plantarum (LP) C88 can prevent liver injury in mice induced by a atoxin B1 [11]; and LP-DSMZ 2017 protects the liver through antioxidant effects [12]. The protective function of lactic acid bacteria as a functional food on the liver has been con rmed. The main mechanisms of carbon tetrachloride (CCl 4 )-induced acute liver injury in mice are oxidative stress, in ammation, and apoptosis [13]. This is similar to human acute chemical liver injury, so it is widely used in animal models to nd potential treatment strategies [14]. However, oxidative stress is the main pathogenic factor of liver injury induced by CCl 4 . In the process of liver metabolism of CCl 4 , hepatotoxic metabolites and excessive free radicals are produced, which deplete reducing agents such as GSH, inhibit the production of antioxidant enzymes such as SOD and CAT, and induce oxidative stress. At the same time, toxic metabolites and free radicals combine with phospholipid molecules, leading to lipid peroxidation and membrane dysfunction.
In addition, they can also bind to other macromolecules, such as proteins and DNA, which can cause cell damage [15].
Yak yogurt is a fermented milk, which is commonly found in ethnic minority areas on the plateau of China. It contains more protein, essential amino acids, and fats than ordinary milk, so it has extremely high nutritional value [16]. Owing to the unique climate, altitude, and technical conditions of the plateau, the taste of yak yogurt and its microbial community are also very rich [17]. The health function of yak yogurt is closely related to the dominant lactic acid bacteria present in it [18]. Experiments have shown that the dominant genus of bacteria in yak milk is Lactobacillus. Preliminary laboratory studies have shown that LP-HFY05 isolated from natural fermented yak milk can reduce alcoholic liver injury in mice [19]; LP-KSFY02 can prevent d-galactose-induced oxidative senescence in mice [20]; and LP-ZS62 can reduce alcoholic gastric injury in mice though antioxidant mechanism [21].
In order to study the preventive effect of L. plantarum on CCl 4 -induced acute liver injury, LP-HFY15 isolated from yak yogurt sampled in the Hongyuan area of Sichuan province was used to gavage mice in this study. By measuring the corresponding indicators of mouse liver and serum, combined with liver histopathological slices, the degree of prevention of LP-HFY15 on the acute liver injury caused by CCl 4 and its possible mitigation mechanism were explored.

Results
Isolation and identi cation of L. plantarum HFY15 The colony is white, round, translucent, with a smooth and matt surface, and a raised center and neat edges (Fig. 1A). Gram staining showed Gram-positive bacteria, which were mainly short and elongated under the microscope, without branches (Fig. 1B).
Tolerance of arti cial gastric juice and bile salts Resistance to gastric juice and bile salts in the gastrointestinal tract is a prerequisite for the function of probiotics [24]. Therefore, the survival rate of LP-HFY15 under arti cial gastric juice and in vitro growth e ciency under bile salt treatment were determined. As shown in Fig. 2, the survival rate of LP-HFY15 in arti cial gastric juice at pH 3.0 is greater than 90%, and it can maintain good growth activity in 0.3% bile salts.

Weight and liver indicators
The liver organ index is the ratio of the weight of the liver tissue of the mouse to its body weight. As shown in Table 1, the average liver weight and liver index of the normal group are the lowest. After CCl 4 treatment, the mice had the highest average liver weight and liver index. Treatment with LP-HFY15 reduced the liver weight and liver index. The liver index of silymarin group and LDSB group also had a downward trend compared with CCl 4 , but the effect was not as signi cant as that of LP-HFY15. Analysis of mouse serum ALT, AST, and TG The levels of ALT, AST, and TG in mouse serum are shown in Fig. 3. ALT and AST levels are often used as indicators to evaluate liver damage. Compared with the normal group, the levels of ALT, AST, and TG in the CCl 4 -induced group signi cantly increased. Compared with the CCl 4 -induced group, the ALT, AST, and TG levels in the LP-HFY15 group were signi cantly reduced. Silymarin is widely used in the treatment of liver damage as a positive control. The silymarin group and LDSB group also had the same trend. However, the levels of silymarin group and LP-HFY15 group were close to the normal group, and a signi cant difference from the LDSB group was observed.
Analysis of MDA, T-SOD, ROS, GSH and CAT in mouse serum Serum MDA content, SOD, ROS, GSH and CAT activity levels are shown in Fig. 4. MDA, SOD, and CAT are important indicators of oxidation. It could be seen from the gure that MDA and ROS levels in mouse serum in the normal group were the lowest among the ve groups, and SOD, GSH and CAT were the highest among the ve groups. Compared with the normal group, the MDA content and the ROS level of CCl 4 -induced group signi cantly increased, and the activities of SOD, GSH and CAT signi cantly decreased. After the treatment with LP-HFY15, the oxidation index trend was opposite to that of CCl 4induced group, which reduced the content of MDA and the level of ROS, and increased the activity of SOD, GSH and CAT. The silymarin group and the LDSB group showed the same oxidation index trend as the LP-HFY15 group, and the values of the silymarin group and LP-HFY15 group were closer to the normal group.
Analysis of serum IL-6, TNF-α, and IFN-γ levels in mice The serum cytokine levels are shown in Fig. 5. Serum cytokines are often used as indicators of liver in ammation. The levels of IL-6, TNF-α, and IFN-γ in the CCl 4 -induced group were signi cantly higher than those in the normal group. Compared with the CCl 4 -induced group, the LP-HFY15 group, LDSB group, and silymarin group signi cantly reduced the levels of IL-6, TNF-α, and IFN-γ. The silymarin group was closest to the normal group, followed by the LP-HFY15 group. The expression level of LDSB group was signi cantly higher than those of silymarin group and LP-HFY15 group.

Histopathological observation
The H&E stained section of the mouse liver is shown in Fig. 6. In the normal group, the liver cells were intact, the nuclei were neatly arranged, and the liver lobules were clearly structured and evenly distributed.
The CCl 4 -induced group showed severe liver cell degeneration, liver lobule disorder, nuclear condensation, and a large number of in ammatory cell in ltrations. In the silymarin group, LP-HFY15 group, and LDSB group, the liver cell degeneration was reduced, nuclei were arranged more neatly, and in ammatory cells were slightly in ltrated.

Discussion
As a type of edible probiotics, lactic acid bacteria can regulate the imbalance of intestinal microbiota composition by increasing the number of bacteria, improving the intestinal epithelial barrier function, and promoting the production of cytokines to prevent obesity, cancer, liver damage, and other diseases [23]. In recent years, studies have been conducted on the use of lactic acid bacteria to alleviate CCl 4 -induced liver injury [2]. Therefore, the LP-HFY15 strain isolated from the yak yogurt in the Hongyuan area of Sichuan Province was used in this study to explore its protective effect on CCl 4 -induced liver injury.
Lactic acid bacteria must be able to tolerate the acidic environment of the gastrointestinal tract and then reach the intestine through food or oral intake [24]. The bile salts in the small intestine will destroy the cell membrane of the bacteria. Therefore, the lactic acid bacteria must have a certain ability to resist gastric juice and bile salts. Only with good tolerance can it reach the intestine with a higher number of viable bacteria and colonize the intestine to exert its probiotic effect. The results show that the survival rate of LP-HFY15 arti cial gastric juice treatment was 92.1%, and the growth e ciency under 0.3% bile salt was 78.8%. The survival rate of L. fermentum Lee used in the experiment at pH 3.0 in arti cial gastric juice was 87.99%, and the growth e ciency under 0.3% bile salt was 25.31% [25]. In contrast, LP-HFY15 has good resistance to gastric acid and bile salt.
CCl 4 can cause liver damage and liver brosis in mice [26]. Liver histopathology is an important clinical criterion for diagnosing liver injury [27]. Through experiments, it was found that CCl 4 treatment caused obvious swelling of mouse liver cells, irregular nucleus size, and lymphocyte in ltration. After the treatment with LP-HFY15, the condition improved, and the liver cells were slightly swollen. Liver weight and liver indicators are usually used as the indicators of liver injury induced by CCl 4 [28]. The results show that treatment with LP-HFY15 could reduce the liver weight and liver indexes in the CCl 4 -induced group, indicating that LP-HFY15 played a positive role in relieving CCl 4 -induced liver injury.
ALT and AST are indispensable catalysts in the normal functioning of the liver. ALT exists in liver cells, while AST exists in the mitochondria of liver cells. When liver cells are damaged, ALT will enter the blood rst, and when liver cells are severely damaged, ALT will also enter the blood, leading to an increase in the transaminase in the serum of mice [29,30]. Therefore, ALT and AST levels can be used to evaluate the degree of liver damage, and their values are positively correlated with the degree of liver cell damage [31]. Liver damage can cause fat in peripheral adipose tissue to be transported to the liver and accumulate, increasing the content of TG in the liver [32]. The results show that LP-HFY15 can regulate the levels of ALT and AST in the liver of mice, reduce the content of TG in the CCl 4 -induced group, and reduce the damage of CCl 4 to the liver.
Acute liver injury is related to liver oxidative stress [33]. The damage caused by oxidative stress can be mitigated by the enzymatic antioxidant defense system [34]. Therefore, this study evaluated the liver's oxidative stress ability by measuring SOD, CAT, ROS, GSH, and MDA and other antioxidant parameters. SOD and CAT are antioxidant enzymes. SOD can catalyze the disproportionation reaction of superoxide anions and scavenge free radicals, while CAT can eliminate hydrogen peroxide in the body and enhance the role of SOD in scavenging free radicals [35]. Reduced consumption of endogenous antioxidants can increase the sensitivity of liver cells to oxidative stress. Therefore, SOD, CAT, and GSH are often used to evaluate the antioxidant activity in the body [15]. MDA is the nal metabolic product of lipid peroxidation, and it is considered an important marker of oxidative stress [36]. In addition, the defense system of antioxidant enzymes may reduce oxidative stress by reducing ROS [37]. Experiments show that LP-HFY15 can signi cantly increase the activity of SOD, GSH, and CAT in mice and reduce the level of MDA and ROS, thereby reducing the oxidative damage caused by CCl 4 to the liver.
The Nrf2 pathway is an important body's self-defense system that can protect tissues from oxidative stress. The transcription factor Nrf2 is the main regulator of cellular antioxidant defense response, and is related to the endogenous antioxidant system [38]. Nrf2 generally exists in the cytoplasm in an inactive form, maintaining low levels of Nrf2 to regulate gene expression. Under oxidative stress, Nrf2 transfers from the cytoplasm to the nucleus, activating the transcription of a variety of antioxidant and detoxi cation genes, which including HO-1 and NQO1 [39]. HO-1 and NQO1 are the downstream antioxidant proteins of Nrf2. HO-1 has anti-in ammatory, anti-apoptotic and anti-oxidant effects on broblasts, hepatocytes and renal epithelial cells. HO-1 also can reduce the amount of mitochondrial oxidation products by inducing autophagy, thereby protecting the heart [40,41]. NQO1 is mainly located in the cytoplasm, but there are also lower levels in the nucleus. Nrf2 has an antioxidant effect by inducing the expression of NQO1 isoenzymes [42,43]. After evaluating the expression of Nrf2 and its downstream molecules HO-1 and NQO1, we observed that the Nrf2-mediated pathway is inhibited in the CCl 4 -induced group. LP-HFY15 shows a protective effect by up-regulating the expression of these molecules, which means LP-HFY15 plays an anti-oxidant effect in CCl 4 -induced liver injury by activating the Nrf2-mediated pathway. These results indicate that the protection of cells from CCl 4 -induced oxidative stress by LP-HFY15 is related to the regulation of antioxidant enzymes, lipid peroxidation and liver antioxidant gene expression.
In ammatory cytokines play a key role in liver injury [44]. CCl 4 induces liver oxidation and in ammation, releases various in ammatory mediators during oxidative stress injury, and signi cantly increases the levels of serum in ammatory factors IL-6, TNF-α, and IFN-γ in mice.
[36] IL-6 promotes the proliferation and differentiation of T lymphocytes and enhances the body's in ammatory response [33,45]. TNF-α can accumulate in ammatory cells, leading to in ammatory cell in ltration and tissue edema [46]. IFN-γ can increase the sensitivity of liver cells to TNF-α and further damage liver cells [47]. It was experimentally found that LP-HFY15 can signi cantly downregulate the levels of IL-6, TNF-α, and IFN-γ, indicating that LP-HFY15 has a good alleviating effect on the in ammatory response induced by CCl 4 in mice.
Apoptosis is a form of programmed cell death. It is the main mechanism that regulates cell death. It usually occurs during the development or aging. It also occurs as a defense mechanism when cells are damaged or stressed, and the damaged cells are removed in an orderly and effective manner through multigene control [48,49]. The mitochondrial pathway regulated by the Bcl-2 family of proapoptotic and antiapoptotic proteins has been shown to play an important role in CCl 4 -induced apoptosis [5]. Bcl-2 is mainly located on the membrane of intracellular organelles and has an antiapoptotic effect. Bax protein is mainly distributed in the cytoplasm. When the cell receives an apoptosis signal, Bax will migrate from the cytoplasm to the mitochondrial membrane, causing damage to the mitochondrial membrane and promoting apoptosis [50,51]. The downregulation of Bcl-2 and the upregulation of Bax will cause mitochondria to release cytochrome c, thereby activating caspase in the cytoplasm, the most important of which is Caspase-3, which ultimately leads to cell apoptosis [52]. According to the experimental data, LP-HFY15 can signi cantly upregulate the expression of Bcl-2 and downregulate the expression of Bax and Caspase-3, indicating that LP-HFY15 can alleviate the apoptosis of liver cells caused by CCl 4 , and protect the normal physiological functions and procedures of liver cells.

Conclusion
In summary, LP-HFY15 has higher resistance to gastric acid and better bile salt survival ability. It can inhibit the production of proin ammatory factors and improve the liver's anti-in ammatory ability. It can also prevent liver cell apoptosis by inhibiting the expression of proapoptotic genes and promoting the expression of antiapoptosis, and ultimately maintaining the normal morphology of liver tissues and liver cells. Moreover, LP-HFY15 can scavenge-free radicals, regulate the release of antioxidant-related enzymes, control liver fat oxidation, and avoid peroxidation. This study provides a basis for the future development of functional foods related to LP-HFY15 to prevent liver damage induced by chemical poisons.

Separation of strains
The samples used in this experiment were collected from yak yogurt in Hongyuan, Sichuan, China. Then, 1 mL of yogurt was added to 9 mL of sterile normal saline, mixed well, and then diluted gradually. Next, 100 µL of bacterial solution with four dilutions of 10 −4 , 10 −5 , 10 −6 , and 10 −7 was taken, spread, and inoculated on de Man, Rogosa, and Sharpe (MRS) (288130, Becton, Dickinson and Company, NJ, USA) solid medium, and incubated at 37°C for 24-48 h. The morphology of the colony was observed; a suitable colony culture was selected and streak with MRS medium to isolate the pure strain. The pure colonies were inoculated into 5 mL MRS liquid medium, incubated for 16-18 h at 37°C, 1 mL of culture solution was taken and centrifuged at 12000 rpm for 5 min. The supernatant was discarded, and 500 mL sterile normal saline was added. The solution was mixed well, and Gram staining was conducted. Finally, a microscopic examination was conducted.
Identi cation and preservation of strains

Evaluation of the tolerance of LP-HFY15 to the gastrointestinal tract in vitro
First, 0.35 g protease (Nanjing Oddfoni Biological Technology Co., Ltd., Jiangsu, China) was added to 0.2 g NaCl (Chongqing Chuandong Chemical Co., Ltd., Chongqing, China) to prepare 100 mL simulated gastric juice. The pH of the solution was adjusted to 3.0, and then it was ltered and sterilized with a 0.45 µm lter. Then, 5 mL of the activated strain culture solution was taken and centrifuged at 3000 rpm for 10 min. The bacteria was collected and washed twice with sterile normal saline, and then 5 mL of normal saline was added to make a bacterial suspension. Then, 1 mL of the resuspension was inoculated in 9 mL of simulated gastric juice, shaken well, and placed in a 37°C incubator for 3 h, and the number of viable bacteria was measured at 0 h and 3 h. The survival rate was calculated using the following formula: Survival rate (%) = number of viable bacteria at 3 h (CFU/mL)/number of viable bacteria at 0 h (CFU/mL)×100 [2,53].
Then, 2% of the inoculum amount was taken, and the overnight cultured bacteria solution was inoculated in an MRS-THIO medium (MRS medium containing 0.2% sodium thioacetate (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) containing 0.0% and 0.3% bovine bile salt. After incubating for 24 h at 37°C, with a blank medium (uninoculated MRS-THIO medium) as the control, the absorbance at 600 nm was measured with a microplate reader (Thermo Fisher Scienti c, Waltham, MA, USA), and the tolerance to bile salts was calculated using the following formula: Growth e ciency (%)=(Bile salt medium OD 600 )/(Blank medium OD 600 )×100 [54].

Animal experiment design
Fifty 6-week-old male Kunming mice weighing 20 ± 5 g were purchased from the Experimental Animal Center of Chongqing Medical University (No. SYXK 2018-0003). All mice were fed standard feed and water under constant conditions in a light/dark cycle of 12 h at a temperature of 25 ± 2°C. After one week of adaptive feeding, the mice were randomly divided into ve groups: normal group, CCl 4 -induced group (Chengdu Kelon Chemical Reagent Factory, Chengdu, Sichuan, China), silymarin group (Shanghai Yuanye Bio-Technology Co., Ltd. Shanghai, China), LP-HFY15 group, and LDSB group, with 10 mice in each group. The mice in the normal group and CCl 4 -induced group were gavaged with 10 mL/kg saline per day; the mice in the silymarin group were given 50 mg/kg silymarin per day; the mice in the LP-HFY15 group were gavaged with 10 9 CFU/kg LP-HFY15 per day. The mice in the LDSB group were given 10 9 /CFU kg LDSB every day, and the body weight of all the mice was measured and recorded for two weeks. Except for the normal group, the mice in all other groups were intraperitoneally injected with 0.8% CCl 4 (10 mL/kg) on the fourteenth day. After all the mice were fasted for 16 h, the mice were sacri ced (Fig. 8).
The whole blood was centrifuged to separate the serum, and it was frozen and stored at −80°C. The liver was separated and weighed, and then freezed at −80°C or xed with 4% formaldehyde solution. Measurement of serum cytokines IL-6, TNF-α, and IFN-γ levels Serum cytokines such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-a), and interferon-γ (IFN-γ) were detected in the serum using cytokine detection kits obtained from Shanghai Enzyme Link Biotechnology Co., Ltd, Shanghai, China.

Preparation of H&E stained sections of liver tissue
The mouse liver tissue was taken, soaked, and xed with 10% neutral formalin, and then dehydrated in 95% ethanol for 24 h. Then, the tissue was embedded in para n, sectioned, and stained with hematoxylin and eosin (H&E) for histopathological analysis. Histopathological changes were observed under an optical microscope (BX43, Olympus, Tokyo, Japan), and the images were recorded.
Measurement of mRNA expression in mouse liver tissue (qPCR Measurement) First, 50-100 mg of liver tissue was taken and placed in a homogenization tube equipped with small steel balls, and then 1 mL of Trizol reagent was added (Invitrogen, New York, USA) to separate and extract the total RNA from the liver homogenate. The concentration and purity of the total RNA were determined using a micro spectrophotometer (Nano-300, Hangzhou Allsheng Instruments Co., Ltd., Hangzhou, Zhejiang, China). Using the total RNA as a template, cDNA was synthesized by reverse transcription. First, 1 µL of cDNA was added to 2 µL of primers (Table 2), 10 µL of premix, and 7 µL of sterile ultrapure water into an eight-tube tube. Then, the mixture was denatured at 95°C for 3 min, annealed at 60°C for 20 s, and heated at 95°C for 1 min. The whole process was carried out for 40 cycles. Using GAPDH as the internal reference gene, the relative expression of mRNA of each target gene was calculated using the formula 2 −∆∆Ct .

Gene
Forward Sequence Reverse Sequence     Tolerance of L. plantarum HFY15 to arti cial gastric juice and bile salts.   Observation of H&E staining of mouse liver. mice were injected with 10 mg/kg CCl4 on the 14th day; Silymarin group: mice were given 50 mg/kg silymarin every day and injected with 10 mg/kg CCl4 on the 14th day; LP-HFY15 group: mice were treated with 1.0×109 CFU/kg (b.w.) of L. plantarum HFY15 every day and injected with 10 mg/kg CCl4 on the 14th day; LDSB group: mice were treated with 1.0×109 CFU/kg (b.w.) of L. delbruechii subsp. bulgaricus every day and injected with 10 mg/kg CCl4 on the 14th day.