Effect Of SIRT3 On NEFA-induced NF-κB Pathway Activation In Bovine Mammary Epithelial Cells

Introduction: Ketosis and mastitis in dairy cows are the most serious diseases threating modern dairy industry,and they often take place synchronously.Silent information regulator3(SIRT3)is a key molecule in the regulation of cellular energy metabolism and plays an important role in the development of perinatal metabolism-related diseases,but its function in mammary tissue of dairy cows has not been reported.In this study,mammary gland tissue of ketotic cows and bovine mammary epithelial cells(BMEC)were used as experimental materials to study the effect of SIRT3 on inflammatory pathways both in vivo and in vitro,further to provide references for revealing the molecular mechanism of co-occurrence of ketosis and mastitis,and then to lay a theoretical foundation for exploring new targets for their prevention and treatment.Methods:(1)Mammary gland tissue samples were collected from 15 ketotic cows and 15 healthy cows.NF-κB signaling pathway activation and SIRT3 protein expression in samples were determined by Western-blot,and mRNA expression of inflammatory cytokine was measured by qPCR;(2)Breast tissues from healthy Holstein cows in mid-lactation(120 ± 20 DIM)were selected.Primary BMEC were isolated,cultured,and purified.BMEC were further identified by immunofluorescence(IF).(3)BMEC were treated with 0,1.2,2.4,and 4.8 m M BHBA for 48 h.SIRT3 protein expression was studied by Western-blot;(4)BMEC were treated with 0 m M β-hydroxybutyric acid(BHBA)+ 0 m M acetoacetic acid(ACAC),1.2 m M BHBA + 0.4 m M ACAC,2.4 m M BHBA + 0.8 m M ACAC,and 4.8m M BHBA + 1.6 m M ACAC for 48 h.SIRT3 protein expression was studied by Western-blot.(5)BMEC were treated with 0,0.6,1.2,and 2.4 m M non-esterified fatty acids(NEFA)for 24 h.NF-κB signaling pathway activation as well as SIRT3 protein expression were studied by Western-blot,and mRNA expression of inflammatory cytokine was studied by qPCR.(6)BMEC were transfected with SIRT3 overexpression adenovirus(Ad-SIRT3)+NEFA for 24 h.Western-blot was used to study the effect of SIRT3 on NF-κB signaling pathway activation.qPCR was used to study the effect of SIRT3 on inflammatory cytokine mRNA expression.IF was used to detect the effect of SIRT3 on NF-κB P65 nucleation.Results:(1)Compared with healthy cows,the p-NF-κB P65/NF-κB P65 ratio,interleukin-1β(IL-1β)and interleukin-6(IL-6)mRNA expression in mammary gland tissue of ketotic cows were significantly increased(P < 0.01).SIRT3/β-actin ratio was also significantly decreased(P < 0.01).(2)After purification,the cells all highly expressed CK-18,a specific skeleton protein in BMEC,which was confirmed as BMEC;(3)There was no significant difference in the SIRT3/β-actin ratios treated with different concentrations of BHBA compared with the blank control group(P > 0.05);(4)There was no significant difference in SIRT3/β-actin ratio between different concentrations of BHBA + ACAC treatment compared with the blank control group(P >0.05);(5)p-NF-κB P65/NF-κB P65 ratio and IL-1β mRNA expression treated with NEFA increased with the increase of NEFA concentration,showing a linear relationship(Linear P <0.01)but no quadratic correlation(Quadratic P > 0.01).IL-6 mRNA expression increased with the increase of NEFA concentration,showing a linear relationship(Linear P < 0.01)and a quadratic correlation(Quadratic P < 0.01).SIRT3/β-actin ratio decreased with the increase of NEFA concentration,showing a linear relationship(Linear P < 0.01)but no quadratic correlation(Quadratic P > 0.01).(6)There were no significant differences in p-NF-κB P65/NF-κB P65 ratio,SIRT3/β-actin ratio,IL-1β and IL-6 mRNA expression in the vector control group compared with the blank control group(P > 0.05).Compared with the vector control group,the p-NF-κB P65/NF-κB P65 ratio and IL-1β mRNA expression were significantly decreased(P< 0.05).IL-6 mRNA expression was significantly decreased(P < 0.01).SIRT3/β-actin ratio was significantly increased(P < 0.05)in the Ad-SIRT3 group.p-NF-κB P65/NF-κB P65 ratio,IL-1β and IL-6 mRNA expression were significantly increased(P < 0.01).SIRT3/β-actin ratio was significantly decreased(P < 0.01)in the vector + NEFA group.Compared with the vector + NEFA group,p-NF-κB P65/NF-κB P65 ratio,IL-1β and IL-6mRNA expression were significantly decreased(P < 0.01),and the SIRT3/β-actin ratio was significantly increased(P < 0.01)in the Ad-SIRT3 + NEFA group.IF results revealed that the nuclear fluorescence intensity of NF-κB P65 was significantly increased in the vector +NEFA group compared with the vector group(P < 0.01),but it was significantly reduced in the Ad-SIRT3 group(P < 0.01).Compared with the vector + NEFA group,the nuclear fluorescence intensity of NF-κB P65 was significantly reduced in the Ad-SIRT3 + NEFA group(P < 0.01).Conclusion:(1)In the mammary gland tissue of ketotic dairy cows,the mRNA expression of inflammatory cytokines is up-regulated,and NF-κB signaling pathway is activated,but SIRT3 protein expression is significantly decreased;(2)Treatment with different concentrations of BHBA with /without ACAC has no effect on BMEC SIRT3 protein expression;(3)NEFA dose-dependently induce inflammatory cytokine mRNA expression,activated the NF-κB pathway,and decreased SIRT3 protein expression in BMEC;(4)SIRT3 inhibits the NF-κB P65 transportation to the nucleus thereby inhibiting high NEFA-induced NF-κB signaling pathway activation and decreasing inflammatory cytokine mRNA expression.