Proanthocyanidins Protect Against β-Hydroxybutyrate-Induced Oxidative Damage In Bovine Endometrial Cells

Ketosis occurring in dairy cows during transition period is associated with increased ketone body concentration,which caused metabolic disorders of systemic dysfunction.The change of β-hydroxybutyrate(BHBA)concentration in dairy cattle is an important mechanism leading to ketosis,and its blood concentration in ketotic cows is always significantly higher than in nonketotic cows.BHBA concentration in blood is always used for diagnosis of ketosis,which is the gold standard.Studies have shown that BHBA could induce oxidative stress and inflammatory reaction in dairy cows,leading to decreased lactation and frequent postpartum diseases,which seriously affect the reproductive performance of dairy cows.Proanthocyanidis(PCs)is a condensed tannin that is a polyphenolic compound.PC have strong antioxidant capacity,can effectively remove free radicals in the body,and are widely used in food,medicine,cosmetics and other fields.This study aimed to demonstrate a protective effect of PC against BHBA-induced oxidative stress damage in bovine endometrial(BEND)cells by activating the nuclear erythroid2-related factor2-antioxidant response elements(Nrf2/ARE)signaling pathway.Methods: Establish a complete and mature cells culture system.The effects of different concentrations of BHBA and PC on the survival rate of BEND cells were detected by CCK-8 for 6h,12 h,24h and 48 h,and the optimal concentration,optimal action time and the best combination method were determined.Detection of changes in oxidation and antioxidant capacity of BEND cells after successful modeling.The change of m RNA and protein expression levels of Nrf2/ARE signaling pathway by q PCR and Western-blot assay.Results:(1)The results of CCK-8 showed that low concentration of BHBA and PC promoted proliferation of BEND cells and inhibited growth at high concentration of BHBA and PC.Based on the above results,we chose BHBA concentrations of 0.6,1.2 and 2.4 mmol/L and PCs concentration of 10μmol/L for simultaneous addition to BEND cells for 24 h.(2)By the detection of oxidation and antioxidant indexes,PC can inhibit the oxidative stress induced by BHBA in BEND cells,greatly increase the content of GSH,T-AOC and reduce the content of MDA,and increase the antioxidant activity of CAT,GSH-PX and SOD.(3)By detecting the Nrf2 / ARE signaling pathway related factors by q PCR and western-blot,PC can increase the expression of Nrf2 and downstream genes GCLC,HO-1,Mn-SOD,NQO-1 m RNA and protein by activating Nrf2 / ARE pathway,which effectively antagonizes BHBA-induced oxidative damage.Conclusions: In summary,our study confirmed that BHBA could cause oxidative stress in BEND cells and might be the key factor leading to reproductive disorders in ketonic cows,whereas PCs can relieve the oxidative damage by activating the Nrf2 signaling pathway.