Silymarin s0292

Recombinant human TGF-β1 (240-B-002) and PDGF (220-BB) were obtained from R&D system. Rabbit anti-phospho-JNK (#9251),-JNK (#9252),-phospho-p38 (#4511),-p38 (#9212),-phospho-Smad2 (#3108),-phospho-Smad3 (#9520),-Smad2/3 (#5678),-phospho-AKT (#2965),-AKT (#4691) and Rabbit anti-cleaved caspase-3 (#9664) were purchased from Cell Signaling Technology. Rabbit anti-phospho-ERK1/2 (sc-101760),-ERK1/2 (sc-292838), mouse anti-GAPDH (sc-47724), and goat anti-Col1A1 (sc-8784) antibodies were obtained from Santa Cruz Biotechnology. Rat anti-F4/80 (MCA497RT) antibody was obtained from Serotec. Sheep anti-albumin (ab8940) and mouse anti-CD68 (ab955) antibodies were purchased from Abcam. Pyrroloquinoline-quinone (PQQ) (D7783), mouse anti-α-SMA antibody (A2547), N-acetylcysteine (NAC) (A7250) and silymarin (S0292) were purchased from Sigma Aldrich. For in vitro studies, PQQ was used at increasing doses of 0.3 μg/ml, 3 μg/ml and 30 μg/ml if not indicated. NAC served as a positive control and was used at the dose of 800 μg/ml.

Monascin and ankaflavin (purities > 99.9%), provided by SunWay Biotech Co., LTD (Taipei, Taiwan), were extracted and purified from Monascus purpureus NTU 568-fermented rice by following the methods of previous research [45 (link)]. Silymarin (S0292) was purchased from Sigma Chemical Co., LTD (St. Louis, MO, USA). Monoclonal ACC1 antibody (3676), polyclonal AMPKα antibody (2532), monoclonal p-AMPKα antibody (2535), monoclonal ERK1/2 antibody (4695), monoclonal p-ERK1/2 antibody (4370), polyclonal p38-MPAK antibody (9212), and polyclonal p-p38 antibody (9211) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Polyclonal Nrf2 antibody (ab31163) and monoclonal HO-1 antibody (ab13248) were purchased from Abcam (Cambridge, UK). Polyclonal IL-6 antibody (sc-1265), polyclonal IL-1β antibody (sc-7884), monoclonal NF-κB antibody (sc-8008), polyclonal PPARα antibody (sc-9000), polyclonal SREBP-1 antibody (sc-8984), and polyclonal COX-2 antibody (sc-1747) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Monoclonal β-actin antibody (MA5-15739) and polyclonal iNOS antibody (PA3-030A) were purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Polyclonal PPARγ antibody (07-466) and polyclonal TNF-α antibody (AB2148P) were purchased from Millipore (Darmstadt, Germany)

All protocols were designed and prepared before the study. The animals were randomized using list randomizer (https://www.random.org/lists) into four groups (n = 8 rats/ each group): (1) control group, (2) diabetic group, (3) diabetes + silymarin (50 mg/kg), and (4) diabetes + silymarin (100 mg/kg).
The animals of control and diabetic groups orally received vehicle (distilled water). In treatment groups, diabetic rats orally received 50 or 100 mg/kg of silymarin^{19 (link),20 (link)} once daily for 8 weeks^{21 (link),22 (link)}. Silymarin (S0292) was purchased from Sigma Company (USA) and prepared in a vehicle with 1.5 ml/kg distilled water. The doses were selected based on the prior studies that showed that silymarin improved diabetes in STZ-diabetic rats^{20 (link)}. Due to none of the animals dying, 32 rats (n = 8/each group) were included in all further analyses. The study monitored and recorded the body weight and blood glucose level.