Escin suppresses immune cell infiltration and selectively modulates Nrf2/HO-1, TNF-α/JNK, and IL-22/STAT3 signaling pathways in concanavalin A-induced autoimmune hepatitis in mice

Animals

Adult male BALB/c mice (25–30 g) were supplied from Vacsera (Giza, Egypt) and maintained in a controlled setting with tap water and food ad libitum. Animal handling and experiments were reviewed and approved by the research ethics committee of Mansoura University, Mansoura, Egypt (Code number: 2022-46).

Drugs and chemicals

Escin, Con A, N-methyl-2-phenylindole, 5,5′-dithiobis (2-nitrobenzoic acid), and vanadium trichloride were supplied from Sigma (St Louis, MO, USA). N-(1-Naphthyl)-ethylenediaminedihydrochloride, carboxymethyl cellulose (CMC), Tris, acrylamide:bisacrylamide (29:1) 40% solution, sodium dodecyl sulfate, ammonium persulfate, and N,N,N′,N′-tetramethylethylenediamine were purchased from Fisher Chemical (Leicestershire, UK). Bovine serum albumen (BSA) and Tween 20 were purchased from MP Biomedicals (Irvine, CA, USA). All other chemicals were of the highest analytical grade available.

Experimental design

Mice were randomly divided into four groups (6 each): Group I (Control): animals were administered escin vehicle [CMC (0.5%) orally for 4 days] and Con A vehicle [normal saline (0.9%) intravenously (i.v.) in the tail vein 8h] before killing. Group II (Con A): animals received Con A (20 mg/kg) i.v. in the tail vein as 0.2% w/v solution in normal saline (0.9%) for 8h. Group III (escin): animals received escin (10 mg/kg/day) orally for 4 days as 0.1% w/v solution in 0.5% CMC in addition to Con A vehicle as described in Group I. Group IV (Escin+Con A): animals received escin, as described in group III, and Con A, as described in group II, 2h after the last escin dose.

The dose of Con A was chosen based on our preliminary studies and previous mouse studies (Trautwein et al. 1998; Streetz et al. 2001) using Con A. The escin dose was also determined based on previous studies (Du et al. 2016; Zhang et al. 2019) and our pilot study demonstrated that the 10 mg/kg dose achieved the maximum hepatoprotection without induction of liver dysfunction when administered alone.

Sample collection and preparation

Eight hours after Con A injection, mice were anesthetized by thiopental sodium (70 mg/kg, i.p) (Elshal et al. 2021). For hepatic histological and immunohistochemical evaluations, the right median lobe was isolated and fixed in 10% (v/v) formalin in normal saline. To isolate serum samples, cardiac blood was collected via puncturing and centrifuged at 1000g for 10 min (at 4 ℃). Samples of liver homogenate were prepared by homogenizing a portion from the left median lobe of the liver in 20 mM Tris–HCl (containing 1 mM EDTA, pH 7.4), followed by centrifugation at 3000g for 20 min (at 4 ℃) and collection of supernatants. Serum and homogenate samples were stored at − 80 °C for subsequent analysis. Another portion from the left median lobe was kept and stored at − 80 °C for western blot analysis.

Determination of liver function biomarkers

Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) activities were measured in serum as hepatocellular injury biomarkers by biochemical kits (Spectrum Diagnostics, Egypt).

Determination of hepatic necroinflammation and immune cell infiltration

Fixed and paraffinized liver sections (5 μm thick) were dewaxed in xylene, mounted on slides, and stained with hematoxylin–eosin (H&E). The slides were then examined for necroinflammation and immune cell infiltration according to the criteria of the histological activity index (HAI) (Ishak et al. 1995). The scoring of necroinflammation was determined as the summation of: 0–4 for periportal or periseptal interface hepatitis; 0–6 for confluent necrosis; 0–4 for focal inflammation, focal necrosis, and apoptosis; 0–4 for portal inflammation. An average neutrophil count was determined for each group via counting ten fields with the highest aggregates of polymorphonuclear leukocytes with segmented nuclei (Elshal et al. 2019).

Additionally, immunohistochemical expression level of the neutrophil marker Ly6G in liver was evaluated via immunostaining by the avidin–biotin complex method (Guesdon et al. 1979) using anti-mouse Ly6G antibody (Cat. No: 127602; BioLegend, CA, USA) based on the manufacturer’s guidance.

Similarly, CD4+ T cell and monocyte infiltration in liver was also evaluated by immunohistochemical determination of the expression levels of the CD4+ T cells and macrophage marker F4/80 via immunostaining using mouse monoclonal CD4+ T cell antibody (M7310; Dako, Denmark) and anti-mouse F4/80 antibody (Cat. No: 123102; BioLegend, CA, USA) according to the manufacturer’s instructions. Semi-quantitative scoring of positively immunostained samples was assigned as follows: 0 for < 5%, 1+ for 6–24%, 2+ for 25–49%, 3+ for 50–74%, and 4+ for 75–100% positively stained cells (Wang et al. 1999).

Determination of hepatic expression levels of pro-apoptotic markers

Hepatocyte apoptosis was determined by examining the pro-apoptotic markers Bax and active caspase-3 via immunostaining by the avidin–biotin complex method (Guesdon et al. 1979) using anti-Bax and anti-cleaved caspase-3 rabbit polyclonal antibodies (Cat. No: A12009; ABclonal, MA, USA and GB11532; Servicebio, Gent, Belgium, respectively) according to the manufacturer instructions.

Determination of oxidative stress and antioxidant parametersDetermination of hepatic malondialdehyde (MDA) concentration

To estimate the extent of lipid peroxidation, hepatic MDA content was specifically measured using the hydrochloric acid-based medium assay previously described by Gérard-Monnier et al. (1998).

Determination of hepatic total nitrate/nitrite (NOx) content

Total nitrate/nitrite (NOx) content in the liver were determined as indicators for the nitric oxide synthesis pathway, using vanadium (III) to reduce nitrate and the Griess reaction as mentioned formerly (Miranda et al. 2001).

Determination of hepatic Nrf2 and HO-1 expressions

Hepatic Nrf2 and HO-1 expressions were determined immunohistochemically via the avidin–biotin complex method (Guesdon et al. 1979) using the anti-Nrf2 rabbit polyclonal antibody (Cat. No: GB113808; Servicebio, Gent, Belgium) and anti-HO-1 mouse monoclonal antibody (Cat. No: sc-136960; Santa Cruz, Texas, USA) as directed by the manufacturers.

Determination of hepatic NF-κB expression

Hepatic NF-κB p65 expression was determined via immunostaining by the avidin–biotin complex method (Guesdon et al. 1979) using the anti-NF-κB p65 rabbit polyclonal antibody (Cat. No: bs-20159R; Bioss Antibodies, MA, USA) as directed by the manufacturer.

Determination of TNF-α, IL-17A, and IL-22 levels

The concentrations TNF-α, IL-17A, and IL-22 were measured by the ELISA MAX™ Deluxe set (BioLegend, SanDiego, CA, USA) in the liver tissue lysates and sera. To prepare liver lysate samples, liver portions (10%) were homogenized in cold lysis buffer (10 mM Tris pH 7.4, 150 Mm NaCl, 0.5% Triton X-100) supplied with an appropriate amount of Protease Inhibitor Cocktail Set 1 (Calbiochem, USA). Thereafter, centrifugation of samples at 7000g for 10 min at 4 °C was carried out for isolating the supernatants to load in the 96-well plate for ELISA.

Western blotting analysis of p-JNK1 and p-STAT3

Portions of liver weighing 20 mg were lysed in a cold buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate) supplied with the protease inhibitor cocktail. To eliminate cell debris, samples were sonicated and incubated for 30 min on ice before being centrifuged at 10,000g for 20 min at 4 °C. Thereafter, the protein concentration was estimated by the Bradford assay (Miranda et al. 2001). Slab gel (10%) was applied according to Roy and Kumar (2014). After gel polymerization, equivalent sample concentrations (30 µg protein/sample) were loaded in the wells of sodium dodecyl sulfate–polyacrylamide gel and electrophoresis was done at 125 V for about 2 h. Gel was stained by 0.1% Coomassie blue R-250 for 2 h and then a solution of glacial acetic acid, methanol, and water (1:3:6, respectively) was used for distaining. Then, the separated proteins were transferred under electric current to a nylon membrane (GE Healthcare). The blotted membrane was then incubated in blocking solution for 1h at room temperature, followed by incubation overnight at 4 °C with a solution encompassing either anti-beta actin mouse monoclonal antibody (Cat. No: mAbcam8224; abcam, MA, USA), anti-JNK1 (phospho-T183) rabbit polyclonal antibody (1/1000) (Cat. No: ab47337; abcam, MA, USA), or anti-phospho-STAT3 mouse monoclonal antibody (1/1000) (Cat. No: sc-8059, Santa Cruz, Texas, USA]. After three cycles of 5 min wash, the membrane was incubated with a solution containing the appropriate concentration of the secondary antibody for 1h at 25 °C. The chemiluminescence detection was used to identify the antibody-bound protein bands, and the Totallab analysis software (Ver.1.0.1) was used for densitometry analysis (Geldoc-it, UVP, England).

Statistical analysis

The data were analyzed using one-way analysis of variance (ANOVA) test, followed by Tukey–Kramer multiple comparison test as post hoc test and expressed as mean ± SE (n = 6). Kruskal–Wallis by rank and the Dunn’s multiple comparison post hoc tests were used to analyze the scores of necroinflammation and immunohistochemistry. Statistical analysis and graphing were performed by GraphPad Prism V8.01 (GraphPad Software Inc., CA, USA).

留言 (0)

沒有登入
gif