隐丹参酮姜黄素复方对BVDV诱导奶牛卵巢颗粒细胞炎性损伤的影响OA

The aim of this study is to investigate the inhibitory effects of the compound composed of Cryptotanshinone(CRY)-Curcumin(CUR)on damage induced by bovine viral diarrhea virus(BVDV)in bovine ovarian granulosa cells(BOGCs).Healthy mature dairy cattle ovaries were collected,and BOGCs were aspirated and isolated.The TCID50 of BVDV on BOGCs was determined using the Reed-Muench method.The drug treating time point was determined accord-ing to the virus loading in BOGCs infected with 100 TCID50 BVDV at 0,6,12,24,48,72,and 96 hours by one-step probe method of RT-qPCR.The safety concentration range of CRY and CUR on BOGCs was determined respectively by CCK-8,and the optimal ratio of the combination's antiviral activity was evaluated through orthogonal experiments.Test groups were set as follows:control group,BVDV group,and medicine treatment group.The mRNA and protein levels of inflammatory factors(HMGB1,IL-1β,IL-6,and TNF-α)were detected by RT-qPCR and ELISA.The apoptosis of cells were analyzed by flow cytometry and RT-qPCR.Estradiol(E2)and progesterone(P4)were measured by ELISA.The re-sults showed that the isolated BOGCs attached the culture surface after being incubated 3-5 days,and appearing the char-acteristics of ovarian granulosa cells.The logarithmic growth phase of cells occurred at 24 h and peaked at 72 h.The TCID50 of BVDV in BOGCs was 10-4.84/0.1 mL.The copies number of virus in BOGCs reached peak at 24 h and with a lgx value of 4.96 after being infected.The best antiviral effect was observed when CRY and CUR are combined in a 10∶5 ratio.Compared to the control group,BVDV infection significantly upregulated the mRNA and protein levels of inflam-matory factors(HMGB1,IL-1β,IL-6,and TNF-α)in BOGCs(P<0.001),while CRY-CUR treatment not only significantly alleviated these inflammatory responses but also significantly reversed BVDV-induced apoptosis in BOGCs(P<0.001).Compared to the control group,the relative expression and protein levels of ovulation-related genes COX-2 and TGS6 were significantly increased(P<0.001),and the levels of E2 and P4 were reduced in the BVDV group(P<0.001).These ab-normal changes were reversed or inhibited by the drug treatment.In conclusion,the compound composed of CRY-CUR alleviates BVDV-induced inflammation,apoptosis,and steroid hormone secretion disorders in BOGCs,demonstrating it is a potential anti-BVDV infection candidate,and holds promising value for further research and development.