Interleukin 6 (il 6)

The rat livers and AML12 cells were lysed by RIPA buffer, separated by 10% polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membrane. After blocking in 5% Bovine Serum Albumin (BSA), the membrane was incubated with the phospho-JAK2 (Tyr931) (1:1,000, affinity), total JAK2 (1:1,000, proteintech), phospho-STAT3 (Tyr705) (1:1,500, abcam), total STAT3 (1:2000, abcam), IL-6 (1:500, bioworld), IL-18 (1:2000, bioworld), IL-1β (1:500, sino biological), TNFα (1:1,000, sino biological), HIF-1a (1:1,000, Novusbio), HIF-2a (1:1,000, Cell Signaling Technology), VEGFa (1:1,000, Novusbio) primary antibody seperately overnight at 4°C, and then was incubated with HRP conjugated secondary antibody for 1 h at room temperature. Western blots were displayed by advanced enhanced chemiluminescence kit and then semi-quantified by Clinx (Shanghai Qinxiang Scientific Instrument Co., Ltd).

The testicular tissue samples were homogenized, and protein quantifications were performed using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein per specimen were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (0.2 μl or 0.45 μm) (Millipore Corporation, Bedford, MA, USA). The membranes were blocked with 5% fat-free milk for 2 h at room temperature, followed by incubation with the following antibodies at 4°C overnight: nuclear factor like-2 factor (Nrf2; Abcam 1 : 1,000), HO-1 (Abcam 1 : 1,000), quinone oxidoreductase-1 (NQO-1; Abcam 1 : 1,000), NF-κB (CST 1 : 1,000), necrosis factor alpha (TNF-α) (CST 1 : 1,000), interleukin-1 beta (IL-1β) (CST 1 : 1,000), interleukin-6 (IL-6) (CST 1 : 1,000), and beta-actin (Bioworld 1 : 10,000). After washing with Tris-buffered saline and Tween 20 (TBS-T), the blots were incubated with the horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (BioSharp, Technology Inc., China) at room temperature for 2 h. Finally, protein bands were detected using Image Lab Software and analyzed using Image-Pro Plus software. All the experiments were repeated at least 3 times.

STZ was purchased from Sigma (USA). Fasudil hydrochloride injection was purchased from Tianjin Chase Sun Pharmaceutical Co., Ltd. (China). CD68, CD11c, and CD206 antibodies were all purchased from the ProteinTech Group (USA). TNF-α, IL-6, and IL-10 antibodies were all purchased from the Bioworld Technology (USA). Phosphorylated myosin phosphatase target subunit 1 (p-MYPT1), iNOS, and Arg-1 antibodies were all purchased from CST (USA). β-Actin antibodies were purchased from Abcam (UK). The BCA protein concentration determination reagent kit was purchased from Nanjing KeyGen Biotech Co., Ltd. (China).