Superecl plus

Xuanhuang Runtong tablets (batch number: 20200101) were purchased from Hunan Shidai Yangguang Pharmaceutical Co., Ltd. (Yongzhou, China). The voucher specimen is stored in the Chinese medicine storage cabinet of the Institute of Chinese Materia Medica, Hunan Academy of Traditional Chinese Medicine. Loperamide hydrochloride capsules (batch number: KFJ2P0P) were purchased from Xi'an Janssen Pharmaceutical Co., Ltd. (Xi'an, China). The reference substances aloin (batch number: 110787-201808, 92.4%), neohesperidin (batch number: 111857-201804, 99.4%), naringin (batch number: 110722-201805, 91.7%), echinacoside (batch number: 111670-201907, 91.8%), and aloe-epine (batch number: 110795-201007, 98%) were purchased from the China Institute for Food and Drug Control.
Alcian Blue and periodic acid Schiff stains were purchased from Visher Corporation (Changsha, China). Rabbit antimouse AQP3 primary antibodies were purchased from ABclonal (Wuhan, China). Cx43 and TLR5 primary antibodies, HRP goat antimouse IgG, HRP goat antirabbit IgG, and coraLite 488-conjugated affiniPure goat antimouse IgG (H + L) were purchased from Proteintech (Chicago, USA). SuperECL Plus was purchased from Advansta (California, USA). The IL-17A primary antibody was purchased from Abcam (Cambridge, UK). TLR5 and IL-17A primers were purchased from Shanghai Shenggong Biological Engineering Co., Ltd. (Shanghai, China).

Radio-immunoprecipitation assay (RIPA) lysate (P0013B, Beyotime, China) was used to extract total protein from mouse spinal cord tissue. SDS-PAGE was used to isolate proteins. The target protein was transferred to nitrocellulose membrane and sealed with 5% skim milk powder for 90 min. Then, the membranes were incubated with primary antibodies NF-κB (10745-1-AP, 1:2,000, proteintech, USA), IL-1β (ab254360, 1:1,000, abcam, UK), TNF-α (17590-1-AP, 1:1,000, ProteinTech, USA), and β-actin (66009-1-Ig, 1:5,000, ProteinTech, USA) and secondary antibodies HRP anti-mouse (AWS0001a, 1:5,000, Abiowell, China) and HRP anti-rabbit (AWS0002a, 1:5,000, Abiowell, China) at room temperature for 90 min. Finally, the bands were visualized using SuperECL Plus (K-12045-D50, Advansta, USA) and photographed using Chemiscope6100 (CLiNX, China). β-actin was used as an internal reference.

Western blotting was performed as described previously [21 (link)]. Total proteins of tissues and cells were lysed in RIPA buffer (Beyotime, China). The concentration of proteins was determined with BCA (HonorGene, China) method. Equal amounts (40 μg) of proteins were fractionated on 10% SDS-PAGE (Melonepharma, China) and transferred onto a nitrocellulose membrane (Millipore). The membrane was blocked with TBST buffer containing 5% nonfat milk for 2 hours at room temperature and incubated with antibodies against ACE2 (Proteintech, USA, 1 : 3000), Mas receptor (Proteintech, USA, 1 : 1500), OPN (Proteintech, USA, 1 : 3000), SM22 alpha (Proteintech, USA, 1 : 1000), α-SMA (Proteintech, USA, 1 : 2000), ADAM17 (Proteintech, USA, 1 : 500), β-actin (Proteintech, USA, 1 : 5000), or GAPDH (Proteintech, USA, 1 : 3000) overnight at 4°C. After washing in PBST 3 times, the membrane was incubated for 1.5 h at room temperature with horseradish peroxidase- (HRP-) conjugated secondary antibody (Proteintech, USA, 1 : 5000). The protein bands were visualized using a Typhoon scanner after treatment with super ECL Plus (Advansta, USA). β-actin and GAPDH were used as internal controls. The specific bands of target proteins were quantified with the Image Lab software.