Cox 2

LPS and Griess reagent (modified) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse TNF-α ELISA kit and IL-6 ELISA kit were purchased from eBioscience (San Diego, CA, USA). The antibody COX2, iNOS, phos-P65 (Ser536), P65, phos-TBK1, TBK1, phos-AKT (Ser473), AKT, phos-S6K (Thr389), S6K, phos–P38 (Thr180/Tyr182), P38, phos–JNK(Thr183/Tyr185), JNK, GAPDH (Cell Signaling, Danvers, MA, USA) and phos–ERK (Tyr204), ERK (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), HRP-linked anti-mouse and anti-rabbit antibodies were obtained from Santa Cruz. SYBR Select Master Mix for RT-PCR amplification was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Primers for TNF-α, IL-6, iNOS and GAPDH were from Invitrogen Life Technologies (Carlsbad, CA, USA).

Chromatographical acetonitrile was bought from Thermo Fisher Scientific (China). Analytical-grade petroleum ether (PE), methanol, and ethyl acetate were bought from Honeywell (USA). Ultrapure water was obtained from a Milli-Q system (Millipore, USA). The other chemicals were analysis-grade chemicals.
RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Logan, UT, USA). Cell counting kit 8 (CCK-8) was bought from DOJINDO (Japan). Antibodies (including β-actin, p-AMPK, AMPK, p-S6, S6, p-P70S6K, P70S6K, p-p53, p53, and COX-2) were purchased from Cell Signaling Technology (USA); Compound C (an AMPK inhibitor) was bought from Santa Cruz Biotech (USA). Matrix basement membrane was purchased from Corning Co., Ltd. (USA). The apoptosis-Hoechst Staining Kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Crystal violet reagent was purchased from Damao (Tianjin, China). Ki-67 was purchased from Wuhan Servicebio Technology Co., Ltd. The four PMFs obtained from CRCP were used in DMSO and were stored at −20°C and diluted for use.

Oroxylin A was bought from Novartis Pharmaceuticals (Basel, Switzerland) and dissolved in dimethyl sulfoxide (DMSO) as a stock solution. Primary antibodies against CDK2, CyclinE, p27, p-p65, p65, and COX-2 were obtained from Cell Signaling (Beverly, MA, USA). Primary antibodies against E-cad, N-cad, Vimentin, p-IκB, IκB, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA). Secondary antibodies conjugated with horseradish peroxidase were obtained from Santa Cruz Biotechnology. All other reagents were purchased from Sigma-Aldrich (Louis, MO, USA).

Antibodies for p65, p-p65, BDNF, claudin-5, occludin, caludin-1, iNOS, COX-2, and β-actin were purchased from Cell Signaling Technology (Beverly, MA). Enzyme-linked immunosorbent assay (ELISA) kits for cytokines were purchased from Ebioscience (San Diego, CA). 4′,6-Diamidino-2-phenylindole dilactate (DAPI) was purchased from Sigma (St. Louis, MO). QIAamp Fast DNA stool mini kit was purchased from Qiagen (Hilden, Germany). Limulus amoebocyte lysate (LAL) assays was purchased from Cape Cod Inc. (East Falmouth, MA). General anaerobic medium (GAM), BL, and DHL media were from Nissui Pharmaceutical Co. (Tokyo, Japan). MRS medium was purchased from BD (Radnor, PA).

Our recent study has described this method detailly [2 (link)]. The primary antibodies used in this present study included NOX2 (ab129068, 1 : 2000, Abcam, USA), AT1 (381666, 1 : 1000, Zenbio, Chengdu, China), ACE (24743-1-AP, 1 : 1000, proteintech, China), Bax (200958, 1 : 1000, Zenbio, Chengdu, China), MMP13 ((820098, 1 : 1000, Zenbio, Chengdu, China), C-caspase-3 (#9664, 1 : 1000, Cell Signaling Technology, Inc. USA), p53 (sc-393031, 1 : 500, Santa Cruz, Bio. Lnc, USA), Klotho (382164, 1 : 1000, Zenbio, Chengdu, China), Bcl2 (381702,, 1 : 1000, Zenbio, Chengdu, China), COX-2 (#12282, 1 : 1000, Cell Signaling Technology, Inc. USA), MMP-3 (380816, 1 : 1000, Zenbio, Chengdu, China), aggrecan (ab36861, 1 μg/mL, Abcam, USA), iNOS (#20609, 1 : 1000, Cell Signaling Technology, Inc. USA), Nrf2 (221102, 1 : 1000, Zenbio, Chengdu, China), HO-1 (#43966, 1 : 1000, Cell Signaling Technology, Inc. USA), type II collagen (collagen II (1 : 1000, ab34712, Abcam), SOD1 (#37385, 1 : 1000, Cell Signaling Technology, Inc. USA), NLRP3 (381207, 1 : 1000, Zenbio, Chengdu, China), ASC (340097, 1 : 1000, Zenbio, Chengdu, China), and GAPDH (5174, 1 : 1000, Cell Signaling Technology, Inc. USA). The secondary antibodies were purchased from Zenbio (380172, 511103, Zenbio, Chengdu, China).

Hind paw skin was formalin-fixed and embedded in paraffin, and skin of 10 μm thickness was cut and mounted on glass slides, deparaffinized, and rehydrated in graded alcohol solutions. The skin sections were subjected to antigen retrieval by heating the sections to 121°C in 0.1 mol/L of citrate buffer (pH 6.0) in an autoclave for 10 minutes and slowly cooling the sections to room temperature. Furthermore, the sections were incubated for 5 minutes with 3% of H₂O₂ to quench the endogenous peroxidase activity. After the blocking of nonspecific sites with 5% goat serum in a phosphate buffered saline (PBS) for 30 minutes, the sections were incubated overnight at 4°C with a rabbit polyclonal antibody against COX-2 (1:200, Cell Signaling, Danvers, MA), iNOS (1:200, Abcam, Cambridge, MA) and nNOS (1:200, Abcam, Cambridge, MA). The sections were then incubated with a secondary antibody conjugated with horseradish peroxidase for 30 minutes at room temperature. Finally, the slides were incubated in 3,3-diaminobenzidine for 5 minutes before undergoing Mayer’s hematoxylin counterstaining for 60 seconds and being mounted.