Smids are outlined in Table S1.2.Animal studiesSprague awley (SD) rats (male, age: ten weeks, weight: 400 50 g) were obtained from the Laboratory Animal Center of Soochow University. The GC-induced ONFH model was established as follows: LipopolysaccharideYANG et al.3 of(LPS, 40 g/kg) was intraperitoneally injected when every day from day 1 to 3, and MPSS (60 mg/kg) was intramuscularly injected once everyday for the following four consecutive days. Thirty-two SD rats have been randomized into four groups (n = eight): (1) DMSO only (handle group); (two) MP and LPS (model group); (3) model group rats treated with MJN110 (10 mg/kg each day, i.p. injection), exactly where MJN110 was administered 1 h ahead of the first LPS injection (pretreatment group); and (4) model group rats treated with MJN110 (10 mg/kg every day, i.p. injection), exactly where MJN110 was administered 3 h immediately after the last MP injection (posttreatment group). The MJN110 dose used was determined by that reported in prior research.224 The femoral head and lengthy bone samples had been harvested at six weeks right after the establishment from the model. The Ethics Committee in the Initially Affiliated Hospital of Soochow University approved all animal experiments.Highlights 1. The expression of monoacylglycerol lipase (MAGL) in BMSCs was enhanced on glucocorticoids (GC) stimulation. 2. The expression of MAGL positively correlated with all the expression of NADPH oxidase and apoptosis-related proteins. three. MAGL inhibition regulated oxidative pressure in BMSCs by way of the Kelch-like ECH-associated protein 1 (Keap1)/Nuclear issue erythroid 2related factor 2 (Nrf2) pathway. four. Pharmacological blockade of MAGL could confer substantial femoral head protection even when administered right after initiation of GCinduced oxidative strain.2.three Micro-computed tomography scansThe femoral heads of rats have been scanned and analyzed applying high-resolution micro-computed tomography (micro-CT) SkyScan 1176 (Bruker, Aartselaar, Belgium). A pair of specimens was placed inside a micro-CT test tube cup. The scanning parameters had been 70 kV, 141 mA, and 1750 ms, having a spatial resolution of 18 m. The following parameters were analyzed employing the CT Analyzer software (Bruker): bone volume (BV, mm3 ), bone volume fraction (BV/TV, ), trabecular thickness (Tb.Th, mm), and trabecular spacing (Tb.Sp, mm).2.Hematoxylin and eosin stainingThe femoral heads of rats had been immersed in 4 paraformaldehyde for 48 h. Soon after 4 weeks of decalcification in 10 HDAC5 Inhibitor Storage & Stability ethylenediaminetetraacetic acid, the specimens have been dehydrated, paraffin embedded, sliced (6 m), and mounted onto glass slides. Soon after hematoxylin and eosin (H E) staining, the sections had been mounted with neutral resins and observed beneath an AxioCam HRC microscope (Carl Zeiss, Oberkochen, Germany).two.6 two.4 Histological and immunohistochemical analysisThe femoral head samples have been harvested at six weeks after the establishment in the model. Immediately after 48 h of fixation and 4 weeks of decalcification, the femoral head samples have been embedded in paraffin and sectioned. The protein expression of MAGL, NOX1, NOX4, and Nrf2 was evaluated via immunohistochemical evaluation (all antibodies had been obtained from Abcam, Shanghai, China). The sections were conventionally dewaxed, rehydrated, and subjected to antigen retrieval, followed by IL-5 Inhibitor review blocking with horse serum for 30 min. Next, principal antibodies and the corresponding secondary antibodies were added dropwise to the specimens, and the signal was created applying three,3-diaminobenzidine. Ultimately, the sections have been counterstained wit.