Protective Effect Of Mesenchymal Stem Cells(MSCs)-mediated ACE2 Against SARA-induced Mastitis In Goats And Its Mechanism

High levels of Lipopolysaccharide(LPS)in the blood caused by subacute rumen acidosis(SARA)can induce mastitis and reduce milk quality in lactating animals.Angiotensin-converting enzyme 2(ACE2)has shown great potential in anti-inflammatory tissue damage,but less research has been done on mastitis in animals.In this study,we investigated the protective effect of local ACE2 on mammary tissue inflammation and the therapeutic effect of combined Mesenchymal stem cells(MSCs)through in vivo and in vitro experiments in dairy goats and mice and conducted a preliminary investigation of the mechanism of action.The study included the following four aspects.1.Role of ACE2 in SARA-induced mastitis and blood-milk barrier damage in goatsThe role of ACE2 in SARA-induced mastitis in dairy goats was investigated by establishing a SARA-induced mastitis model and analyzing the changes in each component of the mammary gland renin-angiotensin system.Eighteen dairy goats in mid-lactation were randomly divided into the control group(high concentrate:roughage ratio 40:60),high concentrate group(high concentrate:roughage ratio 60:40),and buffering agent group(high concentrate:roughage ratio 60:40+buffering agents).At 20 weeks,the pH of rumen fluid was measured,and rumen fluid,milk vein blood,and mammary tissue were collected,and the levels of LPS,NAGase activity,TNF-α,IL-1β,IL-8,Ang Ⅱ,and Ang 1-7 were measured,and pathological tissue sections were prepared.Western blot assay was performed to detect the protein expression of ACE2,AT1R,ZO-1,Occludin,and Claudin-3.Results:1)The pH of rumen fluid of dairy goats in the high concentrate group decreased extremely significantly(P<0.01)and pH<5.8 persisted for more than 5 h.The goats were in SARA status.The histopathological findings of the mammary showed inflammatory cell infiltration in the alveoli and destruction of the mammary structure;the addition of the buffering agents reversed these abnormal results.2)LPS content in rumen fluid and milk vein blood and TNF-α,IL-1β,IL-8 content,and NAGase activity in mammary tissues of dairy goats were significantly increased in the high concentrate group(P<0.01),while the above indexes were significantly decreased in the buffering agents’ group.3)Ang Ⅱ content and AT1R protein expression were highly significantly up-regulated(P<0.01)and Ang 1-7 content and ACE2,ZO-1,Occludin,and Claudin-3 protein expression were significantly down-regulated(P<0.05)in the mammary tissue of dairy goats in the high concentrate group,with the opposite result after buffering agents feeding.Conclusion:High concentrate feeding can induce SARA in dairy goats for a long time.the concentration of LPS increases in SARA,the Ang Ⅱ/AT1R axis is activated in mammary tissue,while the ACE2/Ang1-7 axis is inhibited and the mammary gland is in a state of inflammation and barrier damage.The addition of complex buffers alleviated SARA,ACE2 protein expression was upregulated,Ang Ⅱ content was reduced,and the state of mammary inflammation and barrier damage was alleviated.Suggestion:ACE2 has a protective effect against SARA-induced mammary inflammation and barrier damage through degradation of Ang Ⅱ.2.Cloning of the promoter region of the ACE2 gene in dairy goats and the potential effect of LPS on its transcriptional activityThe study investigated the effect of cloning the promoter region of the ACE2 gene in dairy goats,constructing different lengths of dual-luciferase reporter plasmids and transfecting them into 293T cells,and treating the cells with different concentrations of LPS,Ang Ⅱ,and DIZE(ACE2 activator)to investigate their effect on the promoter activity of ACE2 gene.The experiment amplified the ACE2 gene promoter region(2077 bp)using dairy goat mammary gland genomic DNA as a template and used this as a template to amplify five promoter regions of different lengths,with sizes of 2077 bp,1569 bp,1217 bp,869 bp,and 477 bp,respectively.The five promoter region fragments obtained were later ligated to the pGL3-basic plasmid and transfected into 293T cells after PCR identification of the bacterial solution,enzyme digestion identification,and sequencing analysis;and treated with LPS,Ang Ⅱ and DIZE to detect their effects on the promoter activity of ACE2 gene.Results:1)The ACE2 gene promoter region of dairy goats were successfully cloned with a size of 2077 bp,and multiple transcription factor binding sites and related transcription factors were predicted by bioinformatics analysis in the ACE2 gene promoter region.2)Five dual-luciferase reporter plasmids of different lengths,named PGL3-A1,PGL3-A2,PGL3-A3,PGL3-A4,and PGL3-A5,were constructed.3)DIZE treatment of 293T cells transfected with the above plasmids was found to significantly enhance the promoter activity and promote the transcriptional expression of ACE2 gene,while both low and high concentrations of LPS and Ang Ⅱ significantly inhibited the promoter activity and suppressed the transcriptional expression of ACE2 gene.Conclusion:In this experiment,we successfully cloned the promoter region of ACE2 gene in dairy goats for the first time;we confirmed that high concentration of LPS reduced the transcriptional expression of ACE2 by inhibiting the promoter activity of ACE2,and the degradation of Ang Ⅱ by low-expressed ACE2 resulted in high Ang Ⅱ concentration,and the excessive level of Ang Ⅱ affected the transcriptional expression of ACE2 and aggravated the mammary gland damage.3.The inhibitory effect of MSCs-mediated ACE2 overexpression on LPS-induced mammary epithelial cell injury and its mechanismThe study in the previous chapter found that ACE2 expression was negatively correlated with mastitis in goats,suggesting that activation or overexpression of ACE2 through endogenous activation may be beneficial in ameliorating inflammatory damage in the mammary gland.In this chapter,MSCs were selected to mediate the overexpression of ACE2 to investigate its therapeutic effect on inflammatory damage of mammary epithelial cells and related mechanisms.Mouse mammary epithelial cells(EpH4-Ev)were selected for the experiment,and the mammary epithelial cell injury model was constructed with LPS,while the MSC-ACE2 cell line was constructed with rat bone marrow mesenchymal stem cells(BM-MSCs).EpH4-Ev group(control group),LPS+MSC group,LPS+MSC-GFP group,and LPS+MSC-ACE2 group were established.The following experiments were performed.1)Construct eukaryotic plasmid pVAXl-ACE2 for ACE2 overexpression and lentiviral packaging to screen MSC cell lines that can stably overexpress ACE2.2)To construct a co-culture model of mammary epithelial cells and MSCs and to detect Ang Ⅱ and Ang 1-7 contents and NAGase activity in cell supernatants after LPS treatment of mammary epithelial cells.3)Detection of TNF-α,IL-1β,IL-6,and iNOS expression in mammary epithelial cells.4)Western blot assay to detect the protein expression of cell proliferation and apoptosis-related proteins and key proteins of MAPK/NF-κB signaling pathway and tight junction proteins ZO-1,Claudin-1,and Claudin-2.Results:1)An MSC cell line overexpressing ACE2 was successfully constructed,and the ACE2 gene was still stably overexpressed after multiple passages.2)Compared with the LPS-treated group,Ang Ⅱ content,NAGase activity,and the expression of TNF-α,IL-1β,IL-6,and iNOS were significantly decreased,Ang 1-7 content was increased,protein expression of PCNA and Bcl2 was significantly upregulated,and protein expression of Bax and Caspase-3 was significantly downregulated in MSC and MSC-ACE2 intervention groups.3)The LPS-treated group significantly upregulated the phosphorylation levels of p65,p38,ERK,and JNK(P<0.05)and ZO-1,Claudin-1,and Claudin-2 protein expression(P<0.05)and downregulated the protein expression of IL-10,p-STAT3 and SOCS3 in mammary epithelial cells,and the results after MSC and MSC-ACE2 intervention in contrast.The results suggest that MSCs mediated ACE2 had strong anti-inflammatory and anti-injury effects on LPS-induced inflammatory injury in EpH4-Ev cells by a mechanism that reduced the release of inflammatory factors and promoted blood-milk barrier repair through downregulation of MAPK/NF-κB and upregulation of IL-10/STAT3/SOCS3 pathways,and that the anti-inflammatory and anti-injury effects of MSC-ACE2 were superior to those of MSC alone.4.Therapeutic effect of MSCs combined with ACE2 on LPS-induced mastitis in mice and anti-inflammatory mechanismThe previous chapter demonstrated in vitro that MSCs-mediated ACE2 overexpression can resist mammary epithelial cell damage,and the combination of MSCs and ACE2 was found to have better protective and therapeutic effects.This chapter of the study further clarifies the therapeutic effect of MSCs combined with ACE2 on mammary gland inflammation through in vivo trials.Mice in mid-lactation were selected for the experiment,and mastitis models were established by udder infusion of LPS,while normal control,LPS+MSC-GFP,and LPS+MSC-ACE2 groups were established for the following tests.1)Hi stop athol ogi cal analysis of the mammary gland;2)Determination of NAGase and MPO enzyme activities,various inflammatory cytokines,and Ang Ⅱ and Ang 1-7 content in mammary gland tissue.3)Detection of phosphorylation levels of key proteins of NF-κB and MAPK signaling pathways and expression of blood-milk barrier-related proteins in mammary gland tissue.Results:1)TNF-α,IL-1β,and IL-6 levels and NAGase and MPO activities in breast tissue were significantly increased after LPS infusion in the breast(P<0.05);significant inflammatory pathological changes occurred in mammary gland tissue.After MSC-GFP and MSC-ACE2 treatment,the above indexes were significantly decreased and histopathological damage was reduced,and the effect of MSC-ACE2 was better.2)Compared with the LPS group,Ang Ⅱ content was significantly downregulated,Ang 1-7 content was significantly upregulated,and TLR4 and MyD88 protein expression levels and phosphorylation levels of p65,ERK,JNK,and p38 were significantly downregulated in mammary gland tissues of the LPS+MSC-GFP group and LPS+MSC-ACE2 group(P<0.05).The protein expression levels of ZO-1,Occludin,and Claudin-3 were significantly upregulated in mammary gland tissue(P<0.05),and MSC-ACE2 was superior to MSC-GFP in all cases.The above results suggest that MSCs combined with ACE2 can improve LPS-induced mammary gland inflammatory injury and blood-milk barrier disruption in mice.MSCs combined with ACE2 can significantly reduce LPS-induced mammary gland tissue injury,and its therapeutic effect is better than that of MSCs treatment alone.In this study,we confirmed the protective role of ACE2 during the development of SARA-induced mastitis in goats and demonstrated for the first time that LPS and Ang Ⅱ inhibit the transcriptional expression of the ACE2 gene by suppressing its promoter activity.We established the ACE2 combined with MSCs stable expression cell system and verified its therapeutic effect by in vivo and vitro tests,which proved that the synergistic treatment of ACE2 and MSCs is more effective than using them alone,and provided a new theoretical basis for the use of ACE2 combined with MSCs in the treatment of mastitis.