Protective Effects Of Isochlorogenic Acid A On Mitochondrial Dynamics Imbalance And RLR Injury Induced By Mycotoxins In PAM Cells

Aflatoxin B1(AFB1),deoxynivalenol(DON)and ochratoxin A(OTA)are seriously mixed in feed and raw materials and cause serious harm to animal and human health.Mycotoxins can induce cellular mitochondrial damage by inducing cellular oxidative stress,which in turn produces immunotoxicity.Chlorogenic acid has been shown to have anti-fungal growth,enhancing antioxidant and immune functions in the body.However,the association between mycotoxin-induced mitochondrial dynamic imbalance and RLR pathway damage by chlorogenic acid has not been reported.In this study,we first selected the drugs with the best antagonistic effect against combined mycotoxins from chlorogenic acid,isochlorogenic acid A,and neochlorogenic acid,and then explored the protective effect of this drug on mitochondrial dynamics and RLR pathway disruption in porcine alveolar macrophages(PAM)induced by combined mycotoxins.It provides a theoretical basis for the prevention and control of combined mycotoxin contamination in clinical production and the application of chlorogenic acid.Specific test contents are as follows:Experiment 1: Chlorogenic acid,isochlorogenic acid A,neochlorogenic acid on AFB1,DON,OTA combined exposure of the best drug and concentration screeningIn this study,we first screened the chlorogenic acid isomers with the best effect against combined mycotoxins.CCK-8 assay was used to detect the effect of three chlorogenic acid isomers(2,4,8,16,32,64,128 μg/m L)on the cytotoxicity of PAM cells.The results showed that the isomers of the three chlorogenic acids at 128 μg/m L clearly exhibited cytotoxicity,and the viability of PAM cells remained above 85% at concentrations of 64 μg/m L.Then three chlorogenic acid isomers at low,medium,and high(16 μg/m L,32 μg/m L,and 64 μg/m L)were co-treated with AFB1,DON,and OTA(0.94μg/m L,0.185 μg/m L,and 1.255 μg/m L)combined with mycotoxins for 24 h to detect PAM cell viability.The results showed that the viability of PAM cells was the highest after adding 64 μg/m L of the three chlorogenic acid isomers compared with the combined mycotoxin group alone,indicating that 64 μg/m L of the three chlorogenic acids had the best anti-combined mycotoxin effect.PAM cells were then co-treated with 64 μg/m L of three isomers of chlorogenic acid and combined mycotoxins for 24 h.Cellular ROS levels and GPX4 expression were measured,and 64 μg/m L isochlorogenic acid A was found to extremely significantly reduce combined mycotoxin-induced ROS levels and increase GPX4 expression compared with the other two chlorogenic acids(P < 0.05).Therefore,64μg/m L isochlorogenic acid A was selected for subsequent assays.Experiment 2: Effect of isochlorogenic acid A on mitochondrial dysfunction induced by mycotoxin in PAM cells.Control group(C group),combined mycotoxin group(M group),isochlorogenic acid A group(ICCA group),isochlorogenic acid A and combined mycotoxin co-treatment group(ICCA + M group)were set up.(1)Mitochondrial ROS levels in PAM cells were measured by flow cytometry.It was found that PAM cells treated with combined mycotoxin for 24 h significantly increased the level of mitochondrial ROS compared with group C(P < 0.05).Mitochondrial ROS levels were significantly decreased by approximately 40% in the group supplemented with both mycotoxin and isochlorogenic acid A compared with M(P < 0.05).In summary,isochlorogenic acid A was effective in alleviating mitochondrial ROS elevation induced by combined mycotoxins.(2)The m RNA and protein expression of mitochondrial kinetic-related proteins Drp1,Opa1,Fis1,Mfn1,and Mfn2 were detected by q PCR and WB.The results showed that compared with group C,the expression levels of mitochondrial fusion proteins Opa1,Mfn1 and Mfn2 in PAM cells treated with combined mycotoxins for 24 h were decreased(P <0.05 or 0.01);the expression levels of m RNA and protein of mitochondrial split proteins Drp1 and Fis1 were increased(P < 0.05 or 0.01).Compared with M group,the m RNA level of PAM fusion protein in ICCA + M group was increased(P < 0.05 or 0.01),and the m RNA and protein expression of split protein were significantly decreased(P < 0.01).showed that isochlorogenic acid A was effective in ameliorating mitochondrial dynamics imbalance induced by combined mycotoxins.Experiment 3: Effect of isochlorogenic acid A on innate immune function of combined mycotoxin induced PAM cells.The test groups are the same as Experiment 2.(1)PAM cells were co-treated with 64 μg/m L isochlorogenic acid A and combined mycotoxins(AFB1 0.94 μg/m L,DON 0.185 μg/m L,OTA 1.255 μg/m L)for 24 h.The gene and protein expressions of RIG-I,MAVS,IRF3,and NF-κB were detected.The results showed that combined mycotoxin infection could significantly increase the m RNA and protein expression of RIG-I,MAVS and NF-κB(P < 0.05 or 0.01)and decrease the m RNA and protein expression of IRF3(P < 0.01)in PAM cells.After treatment with isochlorogenic acid A,the gene and protein expressions of RIG-I,MAVS and NF-κB in the cells were significantly decreased(P < 0.05 or 0.01),and the gene and protein expressions of IRF3 were restored(P < 0.01).(2)We then used q PCR and ELISA to detect cytokines downstream of the RLR pathway in cells.The results showed that the m RNA and protein expressions of IL-1β,IL-6,IL-12 and TNF-α were increased(P < 0.05 or 0.01,P > 0.05),and the IFN-α protein level was extremely significantly decreased(P < 0.01)after combined mycotoxin infection.Compared with M group,ICCA + M group significantly decreased the m RNA and protein expression levels of intracellular pro-inflammatory cytokines and increased the m RNA and protein expression of IFN-α.showed that isochlorogenic acid A could ameliorate the inflammatory response induced by combined mycotoxins and regulate the immune function of the body.Conclusion:(1)Chlorogenic acid,isochlorogenic acid A and neochlorogenic acid at64 μg/m L can antagonize the increase of ROS and the decrease of GPX4 expression induced by mycotoxin,and 64 μg/m L isochlorogenic acid A is selected for subsequent tests.(2)Combined exposure to mycotoxins can increase mitochondrial ROS levels and imbalance mitochondrial dynamics in PAM cells.Isochlorogenic acid A at 64 μg/m L was able to ameliorate combined mycotoxin-induced mitochondrial damage,reduce mitochondrial ROS release,and balance the expression of mitochondrial split fusion proteins.(3)Isochlorogenic acid A can effectively improve the activation of RLR pathway induced by combined mycotoxins,reduce the expression of RIG-I,MAVS,IRF3,NF-κB and downstream cytokines,and increase the expression of IRF3 and IFN-α.showed that isochlorogenic acid A could inhibit mycotoxin-induced inflammatory response and improve immune function.In summary,isochlorogenic acid A is expected to be used for the prevention and control of mycotoxins in clinical practice.