P jnk

After incubation with PBS, OA-RD17 (1 nM), lipopolysaccharide (LPS, 1 μg/mL) (Solarbio, China), specific TLR4 inhibitor (1 μg/mL), miR-632 mimic (50 nM), or miR-632 inhibitor (100 nM) for 24 h, respectively, cell lysates (RIPA: PMSF: phosphatase inhibitor = 100:1:1; RIPA and PMSF, Meilun Biotechnology, Dalian, China; phosphatase inhibitor, Roche, Shanghai, China) were used to extract total protein in keratinocytes and macrophages. Moreover, cell lysates were also used to extract total protein in wound tissues from SD rats. The extracted proteins were quantified using the Bradford method (BCA protein analysis kit, Meilun, Dalian, China). The protein samples were then separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electro-imprinted on polyvinylidene fluoride membranes, and recorded and analyzed quantitatively using the Bole exposure software system. Primary antibodies, including GAPDH, Lamin B1, P38, P-P38, ERK, P-ERK, JNK, P-JNK, IκB, P-IκB, P65, P-P65 (Affinity, China), GSK3β, β-catenin, Cyclin D1, c-MYC, and Vimentin (ZEN BIO, China) were used following the provided instructions.

Dextran Sulfate Sodium Salt (DSS) (Cat#9011-18-1) was purchased from MP Biomedicals (Irvine, CA). Specific antibodies including ZO-1 (1:1000; #AF5145; RRID:AB_2837631), Occludin (1:1000; #DF7504; RRID:AB_2841004), Claudin-3 (1:1000; #AF0129; #RRID:AB_2833313), p65 (1:1000; #BF8005:AB_2846809), p-p65 (1:1000; AB_2834435:AF2006), IKBα (1:1000; #AF5002:AB_2834792), p-IKBα (1:1000; #AB_2834433:AF2002), ERK (1:1000; #AB_2833336:AF0155), p-ERK (1:1000; #AB_2834432:AF1015), JNK (1:1000; #AF6318:AB_2835177), p-JNK (1:1000; #AF3318:AB_2834737), p38 (1:1000; #BF8015:Q16539), p-p38 (1:1000; #AF4001:AB_2835330) and GAPDH (1:1000; #AF7021; AB_2839421) were purchased from Affinity Biosciences (OH, USA). Tumor necrosis factor (TNF)-α (Cat# 430915), interleukin (IL)-1β (Cat# 432615), and interleukin (IL)-6 (Cat# 431307) enzyme-linked immunosorbent assay (ELISA) kits were obtained from Biolegend (San Diego, California, USA). Myeloperoxidas (MPO) (A044-1-1), Superoxide Dismutase (T-SOD) (A001-3-2), Malondialdehyde (MDA) (A003-1-2), Glutathione Peroxidase (GSH-PX) (A005-1-2), and Catalase (CAT) (A007-1-1) assay kit was bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

BA (purity over 90%) was purchased from Nanjing Zelang Biotechnology Co., Ltd. (Nanjing, China). The BA standard agent (No. MUST-19010408, purity over 98%) was obtained from Chengdu Man Site Biotechnology Co., Ltd. (Chengdu, China). The Rutin (RU) standard agent (No. L-001-181216) was purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, China). Cholesterol (No. B80859) and soybean lecithin (No. SY-S1-190601) purchased from A.V.T (shanghai, China) Pharmaceutical Co., Ltd. (Shanghai, China). Lipopolysaccharide (LPS) L2880 was obtained from Sigma-Aldrich Co., Ltd. (Missouri, USA). BCA protein analysis kit (No. P0012S) was obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Mouse TNF-α ELISA kit (96T, NO. 21I333) and Mouse IL-1β ELISA kit (96T, NO21T361) were purchased from Excell Bio Co., Ltd. (Shanghai, China). Primary antibodies against TLR4, p-JNK, and p-ERK were purchased from Affinity Biosciences, OH, USA. p-p65 (phospho S536), p65, ERK, and β-actin were all from Abcam (Cambridge, Britain). JNK was obtained from Proteintech Group, Inc. (Chicago, USA). HRP-Goat anti Rabbit and HRP-Goat anti mouse were both from Abcam (Cambridge, Britain). Water was ultrapure water, methanol and acetonitrile were chromatographic grade, and other reagents were analytical grade.

SDS-PAGE and western blots were performed using standard protocols. Antibody binding to bands was detected using an ECL detection system (Sage Creation). The primary antibodies used were specific to MUC17 (Sigma-Aldrich, St Louis, MO, USA), IL-1β (Bioworld, St Louis Park, MN, USA), IL-8 (Bioworld), cdx1 (Abcam), pJNK (Affinity biosciences, Cincinnati, OH, USA), pERK (Santa Cruz, Dallas, TX, USA), p38 (Cell Signaling Technology, Danvers, MA, USA), pp38 (Cell Signaling Technology), PPARγ (Affinity biosciences), pp65 (ZSGB-Bio, Beijing, China), p65 (ZSGB-Bio), p21^waf (Proteintech, Rosemont, IL, USA), p53 (ZSGB-Bio), Myh9 (Abcam, Cambridge, UK), and Actin (Sigma-Aldrich).

RIPA Buffer (Tris–HCl 25 mM pH 7.4, NaCl 150 mM, 1% Triton X-100, Sodium Deoxycholate 1%, SDS 0.1%, EDTA 2 mM) containing a protease/phosphatase inhibitor mixture (Roche) was utlized to lyse cells. After electrophoresis with SDS-PAGE, the separated proteins from the gel (50 μg) were transferred on nitrocellulose (NC) membranes, followed by incubation with primary antibodies. Following incubation with appropriate HRP-labeled secondary antibodies, ECL HRP substrate (Beyotime) was employed to detect signals. The primary antibodies used were as follow: α-SMA (55135-1-AP, Proteintech), Collagen I (14695-1-AP, Proteintech), Timp-1 (CSB-PA023560YA01HU, CUSABIO, Wuhan, China), Vimentin (10366-1-AP, Proteintech), Erk (16443-1-AP, Proteintech), p-Erk (sc-81492, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p38 (14064-1-AP, Proteintech), p-p38 (AF4001, Affinity, Changzhou, China), JNK (AF6319, Affinity), p-JNK (AF3318, Affinity), NF-κB (AF5006, Affinity), p-NF-κB (AF2006, Affinity).

After being treated with ox-LDL and co-incubated with or without BB (50, 25, 12.5 μmol/mL) and EG (25, 12.5, 6.25 μmol/mL) for 24 h, cellular proteins were extracted with RIPA solution for 30 min and the protein concentrations were then quantified by a BCA protein assay kit (Beyotime). Equal cellular proteins were firstly separated by 8% SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes in a pre-cooled transfer solution. After blocking with 5% bovine serum protein for 1.5 h, membranes were incubated overnight at 4 °C with the following primary antibodies: ABCG1 and SR-B1 (Abcam, Cambridge, UK), p-P38, P38, p-P65, P65, GAPDH, β-tubulin (Cell Signaling Technology Inc., Danvers, MA, USA), PPARγ, SR-A1, CD36, P-ERK1/2 ERK1/2, P-JNK, JNK, TLR4 (Affinity Biosciences, Zhenjiang, China) at 1:1000 dilution. Then, membranes were incubated with HRP-conjugated secondary antibody (New England Biolabs, Ipswich, MA, USA) at 1:5000 dilution for 1 h. Finally, enhanced chemiluminescence (ECL) solutions were used to enhance the signal of specific proteins [22 (link)].

Proteins (30 µg) were separated using SDS-PAGE gels. Then the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). The PVDF membranes were then blocked with 5% BSA diluted in TBS for 1 h at room temperature. Primary antibodies against CCR1 (Invitrogen), total JNK (Affinity), p-JNK (Affinity), c-Jun (Affinity), p-p38 MAPK (Affinity), total p38 MAPK (Affinity) and β-actin (Affinity) were then added according to the manufacturers' protocols. The samples were agitated at 4 °C overnight. Goat anti-rabbit IgG HRP-conjugated secondary antibodies (ZSGB Bio, China) was added and incubated at room temperature for 2 h. The densitometric analysis was performed using ChemiDoc Touch Imaging System (Bio-Rad Laboratories).