β tubulin

DNALI1 (Abnova, H00007802-B01P, mouse), β-TUBULIN (Affinity, AF7011, Rabbit), AKAP4 (FineTest, FNab00256, Rabbit), GAPDHS (Sigma-Aldrich, HPA042666, Rabbit), DNAH1 (Thermo Fisher, PA5-57826, Rabbit), DNAH12 (Thermo Fisher, PA5-63952, Rabbit), DNAH7 (Bioss, bs-11023R, Rabbit), DNAH10 (Bioss, Bs-11022R, Rabbit). Anti-DRC1 and anti-AKAP3 were gifts from the Liu laboratory [26 (link)] and the Qi Laboratory [27 (link)], respectively.

DRG tissues were harvested, washed, homogenised, and extracted using RIPA lysis buffer (Beyotime Biotechnology Co., Ltd.) containing protease inhibitor. Protein concentrations of lysates were measured by the bicinchoninic acid method (Beyotime Biotechnology Co., Ltd.). Protein samples were electrophoresed in a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk and incubated with the following primary antibodies overnight at 4 °C: TLR4 (1:1000; Affinity Biosciences, Ltd., Cincinnati, OH, USA); MyD88, Keap1 (1:1000; Cell Signalling Technology, USA); NF-κB p65, p-NF-κB (1:1000; Absin Biotechnology Co., Ltd); PI3K, Akt, phospho-Akt(p-Akt) (1:1000; from Cell Signaling Technology, USA); Nrf2 (1:1000; Abcam); HO-1(1:1000, ABclonal Biotechnology Co., Ltd); and β-tubulin (1:3000; Affinity Biosciences, Ltd., Cincinnati, OH, USA). Afterwards, the membranes were washed with PBST, incubated with HRP-conjugated secondary antibodies (Affinity Biosciences, Ltd.), and visualised by enhanced chemiluminescence (Beyotime Biotechnology Co. Ltd.). The results were quantified using the NIH ImageJ software. Catalog numbers of antibodies in the western blotting test are listed in Additional file 1: Table S3.

Sprague Dawley (SD) rats, weighing 200–250 g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (animal license No.: SCXK (Chuan) 2020-030). The multifunctional animal impact equipment has been authorized patent (self-developed, patent number: ZL 2016 1 0347341.5). hUC-MSCs were provided by the Chengdu KangErmei Biological Cell Preparation Center (Number: G01210001). The ELISA kits for serum amylase, lipase, and rat IL-6, IL-10, TGF-β, and TNF-α were purchased from Shanghai Jiaocai Biological Technology Co., Ltd. Bax, Bcl-2, and caspase-3, and β-tubulin antibodies were purchased from Affinity Biosciences (Cincinnati, USA). TUNEL kits was purchased from Roche Group (Switzerland).

RIMVECs were seeded, divided into groups, and treated as described in the in vitro experiment paragraph. Total proteins were extracted using the radio immunoprecipitation assay (RIPA) cell lysis buffer for 10 min, and protein concentration was determined using the bicinchoninic acid (BCA) protein detection kit (Beyotime Biotechnology, China). A standard western blot protocol was performed, and the following primary antibodies were used overnight at 4°C: TLR4 (1:800) (ABclonal, Wuhan, China), NLRP3 (1:1000) (ABclonal), GSDMD (1:1000) (ABclonal), caspase-1 (1:1000) (ABclonal), ASC (1:1000) (ABclonal), NF-κB p65 (1:1000) (Abmart, Shanghai, China), p-NF-κB p65 (1:400) (Abmart), p-I-κB (1:1000) (Abmart), I-κB (1:1000) (Abmart), IL-18 (1:1000) (Affinity Biosciences, Colorado, USA), IL-1β (1:1000) (Affinity Biosciences), claudin-1 (1:1000) (Affinity Biosciences), claudin-2 (1:1000) (Affinity Biosciences), ZO-1 (1:1000) (Affinity Biosciences), and β-tubulin (1:1000) (Affinity Biosciences) used as loading control, followed by the incubation with HRP (Horseradish Peroxidase)-conjugated secondary antibodies (1:5000) (ABclonal) for 1 h at room temperature. The blots were visualized using a Tanon 5200 chemiluminescence imaging system (Shanghai, China) and quantified using the ImageJ software (National Institutes of Mental Health, Bethesda, MD, USA).

Tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) with phenylmethanesulfonyl fluoride (PMSF) (Biosharp, Anhui, China) on ice after grinding with liquid nitrogen. Cells were collected and washed three times with phosphate-buffered saline (PBS) (KeyGEN BioTECH, Nanjing, China) and then lysed in RIPA with cocktail (Cellpro, Suzhou, China) on ice. After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, CA, USA). After blocking with 5% non-fat milk at room temperature for 2 h, the PVDF membranes were incubated with primary antibodies, including SUN5 (Thermo Fisher Scientific, Sunnyvale, CA, USA, 1:1000), β-tubulin (Affinity, Liyang, China, 1:10,000), pERK1/2 (Cell Signaling Technology, Danvers, MA, USA, 1:1000), and Nesprin2 (GeneTex, Irvine, CA, USA, 1:1000) at 4 °C overnight. After washing with TBST three times, horseradish-peroxidase-coupled mouse or rabbit secondary antibodies (Beyotime, Shanghai, China, 1:1000) were used for hybridization at room temperature for 1 h. Signals were visualized using an enhanced chemiluminescent (ECL) kit (Biosharp, Anhui, China), photographed, and measured using the VisionWorks system (Analytik Jena AG, Jena, Germany). The results are representative of at least three independent experiments.