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.
Identification and preservation of strains
LP-HFY15 was identified using the Basic Local Alignment Search Tool (BLAST) in the National Center of Biotechnology Information (NCBI). This strain is currently stored in China General Microbiological Culture Collection Center (CGMCC, Beijing, China); the preservation number of LP-HFY15 is CGMCC No. 16648. Another positive control strain used in this experiment was Lactobacillus delbruechii subsp. bulgaricus (LDSB) (preservation number AB200048); it was purchased from China Center for Type Culture Collection (Wuhan, Hubei, China).
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 filtered and sterilized with a 0.45 µm filter. 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 Scientific, Waltham, MA, USA), and the tolerance to bile salts was calculated using the following formula: Growth efficiency (%)=(Bile salt medium OD600)/(Blank medium OD600)×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 five groups: normal group, CCl4-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 CCl4-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 109 CFU/kg LP-HFY15 per day. The mice in the LDSB group were given 109/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% CCl4 (10 mL/kg) on the fourteenth day. After all the mice were fasted for 16 h, the mice were sacrificed (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 fixed with 4% formaldehyde solution.
Measurement of ALT, AST, TG, MDA, ROS, GSH, T-SOD, and CAT levels in mouse serum
The kits and instructions provided by Nanjing Jiancheng Institute of Biological Engineering (Nanjing, Jiangsu, China) were used to detect alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), malondialdehyde (MDA) levels, reactive oxygen species (ROS), glutathione (GSH), and total superoxide dismutation (T-SOD) and catalase (CAT) activity in serum enzyme.
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 fixed with 10% neutral formalin, and then dehydrated in 95% ethanol for 24 h. Then, the tissue was embedded in paraffin, 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.
Table 2
Gene
|
Forward Sequence
|
Reverse Sequence
|
SOD1
|
5’-AACCAGTTGTGTTGTCAGGAC-3’
|
5′-CCACCATGTTTCTTAGAGTGAGG-3’
|
SOD2
|
5’-CAGACCTGCCTTACGACTATGG-3’
|
5′-CTCGGTGGCGTTGAGATTGTT-3’
|
Nrf2
|
5’-CACATCCAGTCAGAAACCAGTGG-3’
|
5′-GGAATGTCTGCGCCAAAAGCTG-3’
|
NO-1
|
5’-TGCAGGTGATGCTGACAGAGG-3’
|
5′-GGGATGAGCTAGTGCTGATCTGG-3’
|
NQO1
|
5’-CCTGCCATTCTGAAAGGCTGGT-3’
|
5′-GTGGTGATGGAAAGCACTGCCT-3’
|
GSH
|
5’-AGCAGCTGCTGCACTCTACCAG-3’
|
5′-TGAACGCCGTCCGCATCCTCT-3′
|
CAT
|
5’-GGAGGCGGGAACCCAATAG-3’
|
5′-GTGTGCCATCTCGTCAGTGAA-3’
|
Bax
|
5’-GGGCCTTTTTGCTACAG-3’
|
5′-GACACTCGCTCAGCTTC-3’
|
Caspase-3
|
5’-CAGTGGATTCAAAATCC-3’
|
5′-ATATGCCCATTTCAGGA-3’
|
Bcl-2
|
5’-GATGCTGGAGATGCGGA-3’
|
5′-AGACGTCCTGGCAGCCA-3’
|
IL-6
|
5’-ATGAAGTTCCTCTCTGCAA-3’
|
5′-AGTGGTATCCTCTGTGAAG-3’
|
TNF-α
|
5’-ATGGGGGGCTTCCAGAA-3’
|
5′-CCTTTGGGGACCGATCA-3’
|
IFN-γ
|
5’-GCTTTGCAGCTCTTCCTCAT-3’
|
5′-GTCACCATCCTTTTGCCAGT-3’
|
GAPDH
|
5’-TGACCTCAACTACATGGTCTACA-3’′
|
5′-CTTCCCATTCTCGGCCTTG-3’
|
Statistical Analysis
Three measurements of serum and tissue samples were performed in parallel, and then the average value was calculated. SPSS software (SPSS v.25 for Windows, IBM Software Group, Chicago, IL, USA) was used to average and analyze the data. The Duncan multiple range test was used to evaluate the difference between the mean of each group by one-way analysis of variance. Differences with p < 0.05 were considered statistically significant.