Pirfenidone

Rats were administered intraperitoneally with pirfenidone (250 mg/kg) (Selleck Chemicals, Houston, TX, United States) (Shihab et al., 2002 (link); Burghardt et al., 2007 (link)), SIS3 HCl (2.5 mg/kg) (Selleck Chemicals, Houston, TX, United States) (Li et al., 2010 (link); Liu et al., 2018 (link)) and cyclopamine (10 mg/kg) (Cayman Chemical, Ann Arbor, MI, United States) (Alvarez et al., 2011 (link); Yu et al., 2017 (link)) 30 min before ischemia.
Rats were randomly divided into 9 groups: animals received sham operation and an equal volume of DMSO (Sham); rats received MCAO/R and an equal volume of DMSO (I/R); MCAO/R rats were treated with TGF-β2 inhibitor pirfenidone or Smo inhibitor cyclopamine (I/R + Pir, I/R + CYC); MCAO/R rats were treated with 1.5% ISO post-conditioning for 1 h after immediate reperfusion (ISO); MCAO/R rats were treated with pirfenidone, Smad3 inhibitor SIS3 HCl, cyclopamine, pirfenidone combined with cyclopamine before ISO post-conditioning (ISO + Pir, ISO + SIS3, ISO + CYC, and ISO + Pir + CYC, respectively).

Pirfenidone (Selleck, S2907) was dissolved by a vehicle containing 2% DMSO (Sigma, D2650) and 30% polyethylene glycol 300 (PEG 300; Sigma, 90878). Four‐week‐old mice were randomly divided into two groups (n = 11 for each group). Pirfenidone (250 mg kg⁻¹ day⁻¹; Nakazato, Oku, Yamane, Tsuruta & Suzuki, 2002) or vehicle (control) was orally administered daily for 7 days. Then, the gastrocnemius muscle was acquired for protein extraction and immunofluorescence. For satellite cell isolation, the total muscle of the hind leg was used (n = 8). Regeneration assay was also performed. In the same way, 4‐week‐old mice were divided into two groups (n = 9 for each group). Pirfenidone (250 mg kg⁻¹ day⁻¹) or vehicle (control) was orally administered daily. After 1 day of the first Pirfenidone treatment, CTX was injected into gastrocnemius with 100μl of 10μM (Qiu et al., 2016). Then, the gastrocnemius was isolated on Day 2, Day 6, and Day 12 after CTX injection to analyze the tissue morphology.

The pmATII cells were isolated from mice as previously described [8 (link)]. The pmATII cells were seeded in 12 well-tissue culture plates and cultured in DMEM-F12 supplemented with 10% FCS, 2 mM 1-glutamine, 1% penicillin/streptomycin, 3.6 mg/ml glucose and 10 mM HEPES for 24 h to allow attachment. Cells were then cultured for 12 h in fresh 0.1% FCS containing medium. Subsequently, cells were pre-treated with Nintedanib (1 μM) (Selleck, Houston, TX) or Pirfenidone (500 μM) (Selleck, Houston, TX) or respective DMSO control for 48 h.

Ginkgolide B (purity: 98% by HPLC) was supplied by the National Institutes for the Control of Pharmaceutical and Biological Products (Beijing, China). Pirfenidone (Cat number S2907) was obtained from Selleck (Shanghai, China). The drug of Yinxingneizhi Zhusheye (including Ginkgolide B) was obtained from Chengdu Baiyu Pharmaceutical Company. The drug of Pirfenidone capsules was purchased from Beijing Continent Pharmaceuticals Company. Tgfβ1 recombinant protein (Cat number H00007040-P01) was bought from Amyjet Scientific (Wuhan, China). The cell count kit-8 (Cat number CA1210) was bought from Solarbio Life Sciences (Shanghai, China). Rabbit monoclonal to Tgfβ1 (Cat number ab179695) was obtained from Abcam (CA, USA). Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb (Cat number #8828) was applied by Cell Signaling Technology. GAPDH monoclonal antibody (Cat number 60004-1-Ig) was bought from Proteintech (Rosemont, USA).

The human umbilical vein endothelial cell line EA.hy926 was donated by the research group of Professor Qin Ling of the Chinese University of Hong Kong. anti-MMP-2 (Cat. no.40994S), Anti-p-JAK2 (Cat. no.3771S), anti-p-STAT3 (Cat. no.9145S), anti-p-SRC (Cat. no.6943T), anti-p-ERK (Cat. no.4370S), anti-p-p38 (Cat. no.9216S), anti-p-FAK (Cat. no.8556T), anti-p-AKT (Cat. no.9275S), anti-STAT3 (Cat. no.9139), anti-p-SMAD2 (Cat. no.8828S) and HRP-linked secondary antibody (cat. no.#7074) were purchased from CST. CCK-8 (Cat. no.CK04-3000T) was purchased from DOJINDO. Anti-VEGF (Cat. no.ab46154) and Anti-TGF-β (Cat. no.ab217402) antibodies were obtained from Abcam. Inhibitors of STAT3 (WP1066), ERK (Selumetinib), SRC (Dasatinib), FAK (PF-562271), P38 (SB203580), AKT (MK-2206), EGFR (EGFR-IN-12), VEGFR (SU5204), PDGFR (AG1296), as well as Pirfenidone (Cat. no.S2907) were purchased from Selleckchem. Matrigel (Cat. no.354234) and Transwell (Cat. no.3422) were purchased from Corning. TGF-β (Cat. no.10804-HNAC) was Purchased from Sinobiological. anti-MMP-9 (Cat.no.10375-2-AP) and GAPDH (Cat.no.60004-1-Ig) primary Antibodies were obtained from Proteintech (Wuhan, China). Total RNA miniprep kit (Cat. no. AP-MN-MS- -RNA-250) was Purchased from Axygen. PrimeScript™ RT Master Mix (Cat. no.RR036A) and TB Green Premix Ex Taq II (Cat. no.RR820B) were purchased from Takara.

Pirfenidone (PFD; 10 mg/kg; Catalog No. S2907, Selleck Chemicals, Houston, TX, USA) was administered via drinking water. To protect drug stability, the water bottles were shielded from light. The control animals received an equal amount of water under the same conditions. For monitoring, the animals were weighed three times per week. Water consumption was measured daily, and the water was changed on a daily basis. The PFD treatments started on day 14 post-laser injury until samples were collected (day 21, day 35, and day 42 post-laser). Additionally, to investigate the influence of retinal recovery, we terminated the PFD treatment on day 35 post-laser injury and harvested the retina 7 days later on day 42 post-laser. This sequence is defined as “D42 PFD D35” throughout the manuscript.