Cysteine-rich 61(CYR61) alleviates cyclophosphamide-induced proliferation inhibition in ovarian granulosa cells via suppressing NLRP3/caspase1-mediated pyroptosis.

BACKGROUND: We investigated the level of Cysteine-rich 61 (CYR61) in premature ovarian failure as well as its regulatory molecular mechanism in this study. METHODS AND RESULTS: Cyclophosphamide (CTX) was used to induce OGCs (rat ovarian granulosa cells) and rats to establish in vivo and in vitro premature ovarian failure models. H&E staining was used to detect the pathological changes of ovarian histopathology. Si-NLRP3 (NOD-like receptor thermal protein domain associated protein 3, NLRP3) and si-CYR61 were transfected into OGCs using lipofectamine 3000. RT-qPCR and western blot were used to detect the expressions of CYR61 in ovarian tissue and OGCs. It showed that the expression of CYR61 was significantly down-regulated in premature ovarian failure model. Cell viability was detected using a Cell Counting Kit-8 (CCK-8) kit. TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling) staining was used to detect the apoptosis. 5-Ethynyl-2'-deoxyuridine (EdU) and SA-ß-gal (senescence-associated ß-galactosidase) staining were used to assess the proliferation and senescence. The expression of CYR61 in OGCs and ovarian tissues were detected by immunofluorescence and immunohistochemical staining. Overexpression of CYR61 significantly promoted OGCs proliferation and inhibited pyroptosis and apoptosis. Western blot was used to detect the protein expressions of p53 and p21 in OGCs. Flow cytometry was used to detect the pyroptosis. CYR61 overexpression inhibited the expression of NLRP3 and caspase-1 in CTX-induced OGCs according to western blot results. Moreover, we found that CYR61 overexpression down-regulated the protein expressions of p53 and p21 in CTX-induced OGCs. CONCLUSION: CYR61 inhibited CTX-induced OGCs senescence, and the mechanism may be related to the regulation of caspase-1/NLRP3-induced pyroptosis.

Determination of aflatoxins in milk sample with ionic liquid modified magnetic zeolitic imidazolate frameworks.

The ionic liquid (IL) was introduced to the synthesis system of magnetic zeolite imidazolate framework-8 (M/ZIF-8), which was benefit to the formation of binary imidazole and the co-modification of M/ZIF-8. The morphology and textural properties of ILM/ZIF-8 were characterized by SEM, TEM, BET and BJH. The crystal structural shape and size of MZIF-8 was unvaried with the interventional of IL. The ILM/ZIF-8 was applied to the concentration and determination of aflaoxins (AFB1, AFB2, AFG1 and AFG2) in milk samples based on magnetic solid phase extraction (MSPE) coupled with ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The experimental parameters of the MSPE, including amount of ILM/ZIF-8, pH, type and amount of desorption solvent, extraction time and sample volume were investigated by a univariate method and orthogonal screening. The four AFs were concentrated from the 20 mL milk when 90 mg ILM/ZIF-8 was used as magnetic adsorbent. The extraction efficiency of AFs was higher than 80.0% within 15 min. The limits of quantitative and detection were 7.5-26.7 and 2.3-8.1 ng/L, respectively. The proposed method was applied to the determination of milk samples containing trace amounts of AFs and the recoveries ranged from 79.0% to 102.5%, with RSD below 7.7%.