| Oxidative stress on mammary gland is one of the important reason increasing the incidence rate of mammary gland disease and decreasing the performance of milk production and dairy quality.It is important for maintaining healthy and sustainable high yield of dairy cows to explore the mechanism of vitamin A(VA)to alleviate the oxidative stress of BMEC.The preprotective effect of VA on BMEC oxidative stress and its possible mechanism were discussed from Nrf2/GPx1/NF-?B signaling pathway by using diethyl-enetriamine/nitric oxide adduct(DETA/NO)or lipopolysaccharide(LPS)to induce oxidative damage respectively,by inhibitiing IL-1 biological activity using interleukin 1 receptor antagonist(IL-1ra)antagonism of interleukin 1(IL-1)receptor,and by silencing or overexpressing glutathione peroxidase 1(GPx1)gene using small molecule interfering RNA(siRNA)and overexpression technology.This present study was divided into five parts.The experiment 1 was conducted using the signal factor completely randomized design to study the protective effect and its possible mechanism of different doses of VA(0,0.05,0.1,0.2,0.5,1,2,3,4 ?g/mL)on oxidative damage of BMEC induced by DETA/NO.Results showed that NO-induced damage significantly reduced the activities of selenoprotein GPx and thioredoxin reductase(TrxR),superoxide dismutase(SOD)and catalase(CAT)and the total antioxidant enzymes(T-AOC)ability as well as GPx and TrxR gene and protein expression,inhibited Nrf2 signaling pathway,increased malondialdehyde(MDA)and reactive oxygen(ROS)concentrations and phosphorylation levels of inflammatory signaling pathway NF-?B,and enhanced gene expression of downstream inflammatory factors such as IL-1.The VA preprotection group inhibited the corresponding changes of indexes above and increased the protein expression of retinoic acid receptor ?(RAR?),which means the positive effect of VA on oxidative stress in cell is related to the reduction of NO.VA alleviated the oxidative damage induced by NO in a quadratic dose-dependent manner,and VA at 0.2 to 1 ?g/mL showed better effect and the addition of 1 ?g/mL of VA exhibited the best effect.The experiment 2 was conducted to establish LPS-induced oxidative stree model using cell survival and antioxidant parameters.The results showed that the rate of cellproliferation was reduced to 73.20% by 1.0 ?g/mL LPS with an incubation time 6h.The activities of GPx,SOD and CAT were decreased significantly,and the contents of IL-1,NO and MDA were increased significantly.The results indicated that a concentration of 1?g/mL of LPS with an incubation time of 6h resulted in a significant oxidative stress in BMEC,which was used to establish a standard model of oxidative stress induced by LPS.Basing on the experiment 2,the experiment 3 was conducted as the single factor completely randomized design to study the protective effect and its possible mechanism of different doses of VA(0,0.05,0.1,0.2,0.5,1,2,3,4 ?g/mL)on LPS induced oxidative damage of BMEC.The results showed that the oxidative stress induced by LPS significantly increased the NF-?Bp65 gene expression and phosphorylation levels of nuclear factor ?B inhibit protein kinase ?(IKK?),?B inhibit protein ?(I?B?)and NF-?Bp65 and activated the NF-?B signal pathway,increased its downstream gene and protein expression of IL-1? and induced nitric oxide synthase(iNOS)and promoted the excessive formation of NO.The oxidative stress induced by LPS increased the protein expression of Keap1,inhibited the activity of Nrf2 signaling pathway,and reduced the activity of the selenoprotein GPx1 and its gene and protein expression.The addition of VA increased the protein expression of RAR? and significantly inhibited the changes of the above parameters,indicating that VA has a significant inhibitory effect on the activatin of NF-?B pathway and its downstream factor IL-1? expression resulting from LPS induced oxidative damage.It is suggested that the preprotective effect of VA on LPS-induced oxidative stress may inhibit the expression of IL-1 gene,thereby reducing the activity of iNOS and the generation of NO.The protective effect of VA on the oxidative stress induced by LPS showed a quadratic dose-dependent response with increasing VA,and VA at 0.5 to 2 ?g/mL improved the antioxidant capacity significantly,especially the addition of 1 ?g/mL of VA exhibited the best effect.Basing on the experiment 3,the experiment 4 was conducted using the completely randomized design.The bioactivity of IL-1 was inhibited by IL-1ra to explore the mechanism of VA defensing BMEC from NO oxidative stress via the IL-1/NO pathway.The BMEC was randomly divided into eight treatment groups: control group,VA group,LPS group,IL-1ra group,VA+LPS group,VA+IL-1ra group,VA+LPS+IL-1ra group and LPS+IL-1ra group.The results showed that the VA+LPS group or LPS+IL-1ra group could alleviate oxidative damage induced by LPS through inhibiting the phosphorylation levels of IKK?,IkB? and NF-?Bp65,and inhibit NF-?B signaling pathways,decrease gene and protein expression of IL-1? and iNOS and NO production.All of these resultsindicated that the oxidative stress induced by LPS is due to IL-1 induced gene and protein expression of iNOS causing NO excess generation,which menas the IL-1 induced NO excess generation is an important factor to cause cell oxidative stress damage.In addition,the VA preprotection group significantly increased the protein expression of RAR?,enhanced the gene and protein expression of Nrf2,and up-regulated the gene and protein expression of GPx1.It is suggested that the protective effect of VA on LPS-induced cell oxidative damage probably due to improved expression of the downstream antioxidant GPx1 through the Nrf2 signaling pathway,thereby reducing the gene expression of IL-1.Basing on the experiment 4,the experiment 5 was also conducted as the completely randomized design and the BMEC was induced by LPS.BMEC were randomly divided into eight treatment groups: control group,LPS group,VA+LPS group,VA+LPS+GPx1siRNA group,VA+LPS+GPx1OE group(GPx1 overexpression),VA group,LPS+GPx1OE group,VA+GPx1siRNA group.The mechanism of VA alleviating the oxidative stress of BMEC was investigated from Nrf2-GPx1-NF-?B signaling pathway.Results showed that the VA+LPS+ GPx1 siRNA group compared with VA+LPS group,gene and protein expression of GPx1 and TrxR1 were significantly lowered,but the gene expression and protein expression of IL-1?,IL-6,iNOS and the gene expressions of NF-?Bp65 and NF-?Bp50,phosphorylation levels of IkB? and NF-?Bp65related to NF-?B signaling pathway were all significantly increased.That means the addition of VA can not alleviate LPS induced oxidative damage of BMEC because of silenced GPx1,and silent GPx1 gene significantly weakened the inhibition of VA on increased phosphorylation level of IkB? and NF-?Bp65 and the gene and protein expression of IL-1? and iNOS in cells induced by LPS,weakened the protect effect of VA on BMEC oxidatve damage.Compared with VA+LPS group,GPx1 gene and protein expression,protein expressions of TrxR1 and Keap1 were significantly elevated,inflammatory factor IL-1? gene expression,gene and protein expression of iNOS,the gene expressions of NF-?Bp65 and NF-?Bp50,phosphorylation level of IKK? of NF-?B signaling pathway were all significantly reduced,and the protein expression of Nrf2 was decreased significantly in VA+LPS+GPx1OE group.It is suggested that the GPx1 overexpression can protect BMEC from LPS induced damage.These results further demonstrated that VA reduced oxidative stress in cells by activated Nrf2 signaling pathway promoting GPx1 gene expression and by inhibiting NF-?B signaling pathway.It is concluded that the present investigated the mechanism of VA alleviating BMEC oxidative damage by Nrf2/GPx1/NF-?B pathway,and VA enhanced GPx1 gene and protein expression by activating Nrf2 signaling pathways,then inhibited thephosphorylation level of the NF-?B signaling pathways,and reduced the gene and protein expression of IL-1? inducing iNOS expression,and the generated NO excess,eventually protected the cell from oxidative stress damage. |
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