Morroniside

Forty-eight BALB/c mice (half male and half female; body mass 22–28 g) were purchased from the Experimental Animal Center of Lanzhou University (No. SCXK (Gan) 2018-0002). The mice were housed at a temperature of (25 ± 1)°C, relative humidity of 65% ± 10%, and a 12 hr light–dark cycle. All studies were conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals and in compliance with the regulations of the Gansu Provincial Animal Management Committee. All necessary efforts were made to minimize animal suffering and reduce the number of animals used in the experiments.
Lipopolysaccharide and morroniside were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Anti-iNOS, anti-COX2, anti-Arg-1, anti-CD206, anti-JAK2, anti-p-JAK2, anti-STAT3, anti-p-STAT3, and goat anti-rabbit IgG-HRP antibodies were purchased from Abcam (Cambridge, UK). TNF-α, IL-6, and IL-1β ELISA kits were purchased from Shanghai ZCIBIO Technology Co., Ltd. PBS and trypan-blue solution were purchased from Beijing Solarbio Technology Co., Ltd. A bicinchoninic acid assay (BCA)-100 Protein Quantitative Analysis Kit was purchased from Shanghai Biocolor Biotechnology Co., Ltd. RIPA and BCA protein assay kits were purchased from Beyotime, Beijing. ECL was purchased from Affinity, Shanghai. AG490 was purchased from Shanghai Zerun Bio.

A purity of ≥98.0% was detected for morroniside (Yuanye, Shanghai, China) using HPLC. The BMSCs were incubated with various concentrations of morroniside (0, 2, 4, 8, 16, 32, 64, 128, 256, and 512 μM) in 96-well plates at a density of 5 × 10³ cells per well for 48 h before evaluating using a cell count kit (CCK-8; Wako, DOJINDO, Kyushu Island, Japan). To this end, 10 μL of CCK8 solution was administered to each well, followed by the incubation of the plates for an additional 2 h. Microplate readers were used to measure the OD of each well at 450 nm (Bio-Rad, Hercules, CA, USA). Blank samples were prepared from the culture media. The cell viability was calculated as follows: cell viability = [OD (with morroniside) × OD (blank)]/[OD (without morroniside) × OD (blank)]. The highest concentration (512 μM) was found to reduce cell viability. Two concentrations of morroniside (16 and 256 μM) were thus selected for the following experiments.

Morroniside was purchased from Shanghai Yuanye Biotechnology (Shanghai, China). RPMI 1640 medium, fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Thermo Fisher (USA). NOX4 rabbit polyclonal antibody (14347-1), Beclin-1 rabbit polyclonal antibody (11306-1), LC3 rabbit polyclonal antibody (14600-1), Caspase-3 rabbit polyclonal antibody (19677-1), p62 rabbit polyclonal antibody (55274-1), BAX rabbit polyclonal antibody (50599-2), and Bcl-2 rabbit polyclonal antibody (26593-1) were purchased from Proteintech (USA). p22^phox antibody (ab191512) and donkey anti-rabbit IgG H&L (Alexa Fluor 594) pre-adsorbed secondary antibody (ab150068) were purchased from Abcam (UK). mTOR antibody (2972), p-mTOR Ser2448 antibody (2971) were purchased from Cell Signal Technology (CST, USA). LC3β immunofluorescence antibody (sc-271625) were purchased from Santa Cruz (USA). ROS assay kit (KGT010-1) was purchased from Nanjing Kaiji Bio-Technology (Nanjing, China). Annexin V/FITC apoptosis assay kit was purchased from BD (USA).