Molecular Mechanism Of Diminazene Aceturate And Its Derivative Interfering LPS-induced Inflammatory Damage In Bovine Mammary Epithelial Cells

Diminazene aceturate(DA)has been in clinical use for more than 60 years as a treatment for parasitic diseases in livestock due to its proven efficacy.Recent studies have shown that it also has new uses such as antibacterial,anti-inflammatory,ACE2 activation and immunomodulation,and has good therapeutic effects on glaucoma,uveitis and certain autoimmune diseases(rheumatoid arthritis and inflammatory bowel disease).However,the drug has defects such as high toxic side effects,narrow range of use and short half-life.The modification of the structure of the old drug and the discovery of its new use is an effective means to develop new drugs.Therefore,in this study,the derivative DAD3 was obtained by structural modification and derivatization of the parent nucleus of DA through the addition of sulfonyl chloride reactive groups,we also investigated the role and molecular mechanism of derivatives DAD3 and triazamidine to intervene in LPS-induced inflammatory damage in bovine mammary epithelial cells(BMECs),to lay the foundation for revealing the anti-inflammatory mechanism of triazamidine and derivatives DAD3 on LPS-induced inflammatory response in BMECs,to provide a theoretical basis for the new use of old drugs of triazamidine,and to develop new drugs with triazamidine as the parent nucleus.In this study,the derivative DAD3 was synthesized by structural modification and derivatization studies of the parent nucleus of DA through the addition of sulfonamide reactive groups based on the molecular structural characteristics of diminazene aceturate and drug activity relationship(conformational relationship).Using in vitro cultured BMECs,the cytotoxicity of DA and derivative DAD3 on BMECs,the effect of DA and derivative DAD3 on The anti-inflammatory effects of DA and derivative DAD3 on LPS-induced inflammatory responses in BMECs were investigated using BMECs cultured in vitro,as well as the molecular mechanisms by which DA and derivative DAD3 interfere with LPS-induced inflammatory damage in BMECs.It was found that DA at concentrations less than 30 μg/mL and derivative DAD3 at concentrations less than 20μg/mL promoted the proliferation of BMECs and was less toxic to BMECs;the optimal action concentration of DA was 20μg/mL and derivative DAD3 was 15 μg/mL,and the optimal action time was 24 h.In the selected concentration range,both DA(20μg/mL)and derivative DAD3(15μg/mL)significantly inhibited the pro-inflammatory cytokines IL-1β,IL-6,IL-8 and TNF-8 in LPS-induced BMECs.Both DA(20 μg/mL)and derivative DAD3(15μg/mL)significantly inhibited the mRNA expression of pro-inflammatory cytokines IL-1β IL-6,IL-8 and TNF-α,mitochondrial and intracellular ROS production in LPS-induced BMECs(P<0.05).ACE2 was expressed in BMECs and localized to the cytosol and membrane;ACE2 protein and MasR mRNA expression increased and then decreased,ACE,AT1R mRNA and Ang Ⅱ protein expression increased and Ang 1-7 protein expression decreased with increasing LPS concentration;Spearman correlation analysis showed that the expression of TNF-α,IL-1β,IL-6,IL-8,ACE,Ang Ⅱ and AT1R expressions were significantly and positively correlated with the increase of LPS concentration,while ACE2,Ang1-7 and MasR expressions were negatively correlated with the increase of LPS concentration.Both DA and derivative DAD3 activated ACE2,significantly upregulated the expression of ACE2 and Ang-(1-7)protein(P<0.05),and significantly down-regulated the expression of Ang II protein and AT1R mRNA(P<0.05);however,after silencing ACE2 and adding A779,the agonistic effect of DA and derivative DAD3 on ACE2 disappeared,and the expression of ACE2 mRNA and Ang-(1-7)protein expression was significantly reduced(P<0.05)and Ang II protein and AT1R mRNA expression was significantly increased(P<0.05).DA and the derivative DAD3 had no significant effect on the expression of TLR2 and TLR4 proteins in LPS-induced BMECs,but significantly inhibited the phosphorylation of JNK,ERK and IκB-α proteins(P<0.05),and when ACE2 was silenced and A779 was added,the expression levels of pro-inflammatory cytokines IL-1β,IL-6,IL-8 and TNF-α were significantly decreased(P<0.05),and the phosphorylation levels of p38,JNK1/2/3,ERK 1/2 and IKB-αproteins were significantly increased(P<0.05).The results showed that the derivative DAD3,synthesized by adding sulfonyl chloride reactive groups,had good solubility and low toxicity to BMECs.DA and derivative DAD3 had good protective effects against LPS-induced inflammatory injury in BMECs and good elimination of pro-inflammatory cytokine production;it can activate ACE2,promote the expression of ACE2 and Angl-7,and inhibit AngⅡ And ATIR expression.Further analysis of its mechanism of action found that it is mainly through the activation of ACE2/Angl-7/MasR axis to inhibit MAPK(p38,ERK1/2 and JNK1/2/3)and NF-κB(IκBα)pathway protein phosphorylation to exert anti-Inflammatory effect.