| The causes of milk cow mastitis are mostly caused by pathogenic microorganism infection,among which bacterial mastitis accounts for about 90%.Heme oxygenase-1(HO-1),as an antioxidant enzyme,is involved in the regulation of cellular inflammation,apoptosis and autophagy,and there are few reports on its association with mastitis in dairy cows.Lipopolysaccharide(LPS)is the main pathogenic factor of bacterial mastitis,which can reduce the expression of antioxidant enzymes in cells,cause damage and stress of breast tissue,and is an important cause of inflammation and infection.Therefore,LPS is often used to establish inflammatory cell models for in vitro studies.In this paper,from the perspective of HO-1’s involvement in cellular inflammation,apoptosis and autophagy,dairy mammary epithelial(MAC-T)cells were treated with LPS based on the PI3K/mTOR signaling pathway,and cells were treated with exogenous HO-1 and PI3K/mTOR signal blocker LY294002 or Rapamycin.The optimal treatment time and dose of HO-1 were screened by CCK-8,inflammation,autophagy and apoptosis factors were detected by RT-PCR,ELISA and Western blot,the number of autophagic lysosomes was observed by transmission electron microscopy,and apoptosis rate was detected by flow cytometry.To elucidate the effects of HO-1 on inflammation,apoptosis,autophagy and PI3K/mTOR signal of MAC-T cells,and reveal the potential molecular mechanism of HO-1 regulation on inflammation,apoptosis and autophagy of MAC-T cells.The results are as follows:Anti-inflammatory effect of HO-1 inMAC-T cells: The cell viability was determined by CCK-8 method,and the concentration of MAC-T cells treated with HO-1 was 0.06μg/mL for 12 h.MAC-T cells were treated with 100μg/mL LPS for 12 h.mRNA expression and content of related inflammatory factors were detected by RT-PCR and ELISA.The results showed that the mRNA expressions of IL-6,IL-8and TNF-α in cells were significantly up-regulated by LPS,and the contents of IL-6,IL-8 and TNF-α in cell supernatant were significantly increased.The mRNA expressions and contents of IL-6,IL-8 and TNF-α in the group treated with LPS and HO-1 were significantly decreased compared with those in the group,suggesting that HO-1 could alleviate the LPS-induced inflammation of MAC-T cells to a certain extent.Antiapoptotic effect of HO-1 onMAC-T cells: RT-PCR and Western blot results showed that Bax and Caspase-3 mRNA and protein levels inMAC-T cells were significantly up-regulated and Bcl-2 mRNA and protein levels were significantly inhibited by LPS induction.Compared with the LPS group,Bax and Caspase-3mRNA and protein levels showed different decreases and Bcl-2 mRNA and protein levels were significantly increased after co-treatment of LPS with HO-1.Flow cytometry detection of MAC-T cell apoptosis rate showed that LPS treatment caused a significant increase inMAC-T cell apoptosis rate;after combining with HO-1treated cells,the the apoptosis rate of MAC-T cells was significantly reduced.It was suggested that HO-1 played an anti-apoptotic role in inflammatory MAC-T cells.Anti-autophagy effect of HO-1 inMAC-T cells: RT-PCR and Western blot results showed that LC3 B and Beclin1 mRNA levels inMAC-T cells were significantly increased and p62 mRNA expression was significantly down-regulated by LPS treatment,while LPS promoted LC3II/LC3 I ratio and Beclin1 protein expression and p62 protein levels were inhibited.Compared with the LPS-treated group,combined with HO-1 treatment,LC3 B and Beclin1 mRNA levels were differentially suppressed and p62 mRNA levels were significantly up-regulated.LC3II/LC3 I ratio and Beclin1 protein levels were significantly decreased and p62 protein levels were increased.Transmission electron microscopy showed that the number of cellular autophagic lysosomes was significantly increased in the LPS group,and the number of autophagic lysosomes was significantly decreased in the combined HO-1 and LPS treatment group compared with the LPS group.It was suggested that HO-1 exerted an inhibitory effect on autophagy in inflammatory MAC-T cells.PI3K/mTOR signal mediates the anti-inflammatory effect of HO-1: In combination with PI3K/mTOR pathway inhibitor LY294002(20μM)or Rapamycin(10 nM),Western blot results showed that LPS reduced the P-AKT/AKT ratio and inhibited PI3K/mTOR signal.HO-1 alleviates the inhibition effect of LPS on PI3K/mTOR pathway.At the same time,the P-AKT/AKT ratio was up-regulated in the HO-1 group,which activated PI3K/mTOR signal.After blocking PI3 K or mTOR signal,the inhibition ability of HO-1 on LPS-induced inflammatory response was weakened.Compared with the group treated with LPS and HO-1,The expressions of IL-6,IL-8 and TNF-α were up-regulated inMAC-T cells,and the results of ELISA and RT-PCR were similar.These results indicate that the anti-inflammatory effect of HO-1 is realized by activating PI3K/mTOR signal.PI3K/mTOR signal mediates the inhibitory effect of HO-1 on apoptosis: RT-PCR and Western blot results showed that after blocking PI3 K or mTOR signal,the mRNA and protein expressions of Caspase-3 and Bax were increased,and the mRNA and protein levels of Bcl-2 were down-regulated compared with HO-1 and LPS co-treatment group.Flow cytometry showed that the anti-apoptotic effect of HO-1 on inflammatory cells was reversed after treatment with PI3 K or mTOR inhibitors,and the apoptotic rate was significantly increased compared with HO-1 and LPS co-treatment group.It is suggested that HO-1 plays an anti-apoptotic role in inflammatory MAC-T cells depending on PI3K/mTOR signal.PI3K/mTOR signal mediates the anti-autophagy effect of HO-1: RT-PCR and Western blot results showed that after blocking PI3 K or mTOR signal,the anti-autophagy effect of HO-1 was weakened,LC3Ⅱ/Ⅰ ratio was increased,Beclin1 mRNA and protein expression were up-regulated,and p62 mRNA and protein levels were inhibited,compared with HO-1 and LPS co-treated groups.It is suggested that HO-1 plays an anti-autophagy role in inflammatory MAC-T cells depending on PI3K/mTOR signal.In conclusion,HO-1 can improve cell viability and inhibit inflammation,apoptosis and autophagy activities of inflammatory MAC-T cells.HO-1 exerts anti-inflammatory,anti-apoptosis and anti-autophagy effects in inflammatory MAC-T cells depending on PI3K/mTOR signal. |
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