Inhibition Of Epicatechin Gallate In Lps-induced Bovine Mastitis And Pyroptosis And Underlying Molecular Mechanisms

Dairy mastitis is a kind of multiple diseases of dairy cows.This disease is an inflammatory reaction caused by infectious or environmental pathogenic microorganisms and physical factors that stimulate mammary glands to a certain extent.In the inflammatory pathway caused by Escherichia coli infection,Toll-like receptor 4(TLR4)recognizes the stimulus signal of LPS(lipopolysaccharide),Nuclear factor kappa B(NF-κB)pathway and NLRP3(NOD-like receptor 3)inflammatory bodies were activated to cause pyrodeath and aggravate mastitis.Antibiotics are still used to treat mastitis in cattle worldwide.Due to the extensive use of antibiotics,bacterium resistant to antibiotics continue to increase,and antibiotic resudues remain in milk,which lead to the decline of milk quality and the threat to human health.Therefore,it is of great significance to find anti-inflammatory agents in the process of scorched cell death.Natural plant active ingredients can perform antibacterial,anti-inflammatory,antioxidant and anticancer functions through a variety of signaling pathways,and their effects in mastitis have attracted wide attention around the world.Epicatechin gallate(ECG)is a kind of polyphenols,which have a series of pharmacological effects such as anti-oxidation,anti-cancer,anti-inflammation,anti-apoptosis,anti-obesity and neuroprotection.However,the roles of ECG in mastitis have not been reported.In this study,the in vivo and in vitro inflammatory model was established by LPS(lipopolysaccharide)stimulation of bovine mammary epithelial cell lines MAC-T and BALB/c mice.The optimal concentrations of ECG on bovine mammary epithelial cells was screened by MTT method.The expression levels of inflammatory factors(TNF-α,IL-1β,IL-6),oxidative stress factors(COX-2,INOS,MPO)and pyrocytosis factors(ROS,LDH,IL-18)were detected by ELISA and microassay.Inflammation of the breast tissue of BALB/c mice was detected by HE staining(hematoxylin-eosin staining).Inflammatory bodies(NLRP3,caspase-1,ASC)and GSDMD-N in NF-κB pathway(IκB,p65,p-p65,p-IκB),caspase-11,NLRP3(NOD-like receptor 3)were detected by Western blot.The immunofluorescence signals of p65 nuclear translocation,NLRP3 and GSDMD were detected by immunofluorescence method.Cell death was detected by Annexin V-FITC/PI staining and flow cytometry.The results are as follows:1.After the treatment of MAC-T cells with ECG,three concentrations showing noncytotoxic effects on cell viability and proliferation were screened at 1μg/m L,15μg/m L,and 30μg/m L.2.To investigate the anti-inflammatory effects of ECG on mastitis,six treatment groups including blank control(C),LPS,LPS+DEX,LPS+ECG(1 μg/m L,15 μg/m L and 30 μg/m L)were designed.ELISA results showed that: Compared with the blank control,LPS treatment significantly up-regulated expression of inflammatory factors(TNF-α,IL-1β and IL-6),oxidative stress factors(COX-2 and i NOS)(P<0.01)and pyroptosis indicators(ROS,LDH,IL-18)(P<0.01);however,LPS+ECG(1μg/m L,15μg/m L and 30μg/m L)adiminstration significantly down-regulated LPS-induced expression levels of inflammatory factors(TNF-α,IL-1β,IL-6),oxidative stress factors(COX-2,i NOS)and pyroptosis indicators(ROS,LDH,IL-18)in a manner of dose-dependence(P<0.05).Moreover,the expression of MPO(myeloperoxidase),an indicator of leukocyte exudation,was also significantly decreased(P<0.01).3.The results of flow cytometry showed that LPS+ECG(1 μg/m L,15 μg/m L,30μg/m L)treatment could significantly reduce cell death rate compared with LPS treatment(P<0.01).Annexin V and PI staining showed that LPS+ECG(1 μg/m L,15μg/m L,30 μg/m L)treatment obviously relieved cell membrane rupture destroyed by LPS.4.In order to investigate the molecular mechanism of ECG,expression of NF-κB and NLRP3 inflammasome were detected in the cells treated as above.Western blot results showed that LPS+ECG(1 μg/m L,15 μg/m L,30 μg/m L)groups had a significant down-regulation in protein expression of the NF-κB pathway(IκB,p65,p-p65,p-IκB),caspase-11,NLRP3 inflammasome(NLRP3,caspase-1,ASC)and GSDMD-N(P<0.01).Immunofluorescence results also showed that ECG reduced LPS-induced nuclear translocation,NLRP3 and GSDMD-N fluorescence signals.5.To further reveal that ECG inhibited LPS-induced pyroptosis by NLRP3 and Caspase11,MAC-T cells were treated with MCC950(a NLRP3 inhibitor)and AC-FLTD-CMK(a caspase-11 inhibitor),and five treatment groups were designed as control C group,LPS group,LPS+ECG(15 μg/m L),LPS+ECG(15 μg/m L)+MCC950(5 μM),LPS+ECG(15 μg/m L)+AC-FLTD-CMK(2 μM).Western blot analysis showed that MCC950 and AC-FLTD-CMK could significantly reduce the protein levels of NF-κB(p-p65,p-IκB),NLRP3 inflammasome(NLRP3,caspase-1,ASC)and GSDMD-N(P<0.01);and the results were better than ECG alone.6.To validate the anti-inflammatory effect of ECG,MAC-T cells were treated with islicorice(ISL)and epicatechin(EC,similar compounds of ECG,and PCR and ELISA results showed that ISL(2.5,5,10 μg/m L)and EC(1.5、7.5、15、30 μg/m L)significantly decreased the expression levels of LPS-induced inflammatory cytokines TNF-α(P<0.01),IL-1β(P<0.01),IL-6(P<0.05),COX-2(P<0.01),i NOS(P<0.01).The results of Western blot showed that ISL and ECG could significantly inhibit the expression levels of NF-κB phosphorylation induced by LPS(P<0.01).Meanwhile,immunofluorescence results showed that ISL and ECG could effectively inhibit the nuclear translocation of p65.7.To further validate the in vitro results,50μL LPS solution at the concentration of 200μg/m L was injected into the fourth pair of nipples of female mice after 7-10 days of lactation,and ECG of 10,20,40mg/kg was also administrated through intraperitoneal injection for 24 h.HE results showed that compared with the LPS group,ECG at 10,20 and 40 mg/kg significantly reduced the histopathological changes of mammary tissues caused by LPS.ELISA results showed that expression of inflammatory factors(TNF-α,IL-1β,IL-6),oxidative stress factors(COX-2,INOS,MPO)and pyroptosis indicators(ROS,LDH,IL-18)were significantly decreased in 10,20 and 40mg/kg of ECG treatment groups(P<0.01).Western blot results showed that LPS+ECG(10,20,40 mg/kg)groups significantly down-regulated the protein levels of NF-κB(IκB,p65,p-p65,p-IκB),NLRP3 inflammasome(NLRP3,caspase-1,ASC)and GSDMD-N(P<0.01).The in vivo results are consistent with the in vitro findings,indicating that ECG alleviates mastitis by inhibiting the NF-κB pathway and cell pyroptosis.8.A recent study of ours has shown that EC regulates the activity of NF-κB pathway through transmembrane protein 35A(TMEM35A).To determine whether ECG inhibited LPS-induced pyroptosis by TMEM35 A,we used CRISPR/cas9 to knockdown TMEM35 A expression in MAC-T cells.Western blot results showed that knockdown of TMEM35 A significantly increased the levels of p-IκB and p-p65 as well as the protein expressions of TNF-α and i NOS in MAC-T cells treated with LPS(1μg/m L)+ECG(15μg/m L)(P<0.01).However,when transfected with p CMV-TMEM35A-His,the levels of P-iκB and p-p65 and the expressions of TNF-αand i NOS were significantly decreased in MAC-T cells(P<0.01).In conclusion,ECG inhibits LPS-induced inflammation and pyroptosis in MACT cells and the mouse mammary gland through the NF-κB pathway and NLRP3 inflammasome,and TMEM35 A mediates inhibition of ECG in the NF-κB pathway.The results of this study revealed the molecular mechanisms of anti-inflammation and anti-pyroptosis of ECG,and provided a new idea for further prevention and treatment of bovine mastitis.