Study On The Mechanism Of Pomegranate Inhibiting Liver Injury Induced By Staphylococcal Enterotoxin A

This study was funded by the National Natural Science Foundation of China(31772082).Staphylococcal enterotoxin A(SEA)is a common foodborne bacterial toxin that can cause food poisoning.It can cause vomiting,inflammation and other adverse reactions,posing a serious threat to public health.However,the current research on the pathogenesis of SEA mainly focuses on the intestinal tract,while the toxicity to the liver has rarely been reported.Food preservatives are additives that prevent spoilage caused by microorganisms,prolong the shelf life of food,and prevent the reproduction of microorganisms that cause food poisoning.Punicalagin(PUN)is an effective natural antioxidant with anti-inflammatory and bacteriostatic effects,but comparatively little research has been done on its toxin effects.This paper aims to explore the inhibitory effect of PUN on liver injury induced by SEA through oxidative stress and mitochondrial autophagy.The contents of this study will contribute to a more comprehensive understanding of the toxicity of SEA,and also lay the foundation for further development of PUN as a potential food preservative targeting bacterial toxins such as SEA.The main research contents and results are as follows:(1)The effects of SEA on hepatocytes indury through oxidative stress and mitochondrial autophagy were verified.By detecting the levels of reactive oxygen species(ROS)and related protein indexes in human normal hepatocytes HL-7702(L02)after SEA treatment,it was found that the superoxide dismutase(SOD),glutathione(GSH)and catalase(CAT)in L02 hepatocytes decreased by 26.69 ±2.86%,32.20 ± 4.83% and 73.89 ± 5.70%,and malondialdehyde(MDA)increased by45.45 ± 1.70%.At the same time,the Nrf2 pathway was also activated.The morphology of mitochondria in L02 hepatocytes treated by SEA and the changes of related indicators were detected.It is found that SEA could induce mitochondrial membrane potential(ΔψM)significantly decreased,the content of mitochondrial autophagy related proteins was changed,and the co-localization between mitochondria and autophagosomes was increased.The results showed that SEA could induce mitochondrial autophagy in L02 hepatocytes.The addition of NAC indicated that SEA induced mitochondrial damage in L02 hepatocytes by inducing massive production of ROS,leading to an increase in mitochondrial autophagy.In addition,the content of ALT,AST,and apoptosis-related proteins was measured,which confirmed that SEA could induce damage to L02 hepatocytes.(2)The effects of SEA on liver injury in mice through oxidative stress and mitochondrial autophagy were investigated.The liver tissue of mice in the SEA group showed activation of the Nrf2 protein pathway,and the levels of SOD and CAT were significantly reduced to 65.5 ± 0.2% and 56.8 ± 1.1% of those in the control group.At the same time,hematoxylin eosin(HE)staining was used to observe the pathological state of liver indury in mice,which proved that SEA could induce oxidative damage in mice liver.The changes in the content of proteins which related to mitochondrial autophagy in mice liver after SEA treatment revealed that SEA significantly upregulated the protein expressions of LC3,Parkin,Pink1,and Cytc,while P62 was significantly downregulated.It was demonstrated that SEA could induce mitochondrial autophagy in mice liver.The test found that SEA can induce the content of ALT and AST in the serum of mice to increase to about 2 and 1.5 times that of the control group,while the content of apoptosis-related proteins in the liver also significantly changes,indicating that SEA could induce liver injury in mice.In conclusion,SEA induced oxidative stress and mitochondrial autophagy in mice liver,leading to liver indury in mice.(3)It was explored that PUN could inhibit SEA-mediated liver injury via oxidative stress and mitochondrial autophagy.It was found that the PUN of high dose pretreatment(20 μM)could significantly reduce the ROS fluorescence intensity induced by SEA from 1.98 to 0.55,gradually restore oxidative stress related indexes to normal levels,and the Nrf2 protein pathway also tend to the level of control group.The results showed that PUN pretreatment can effectively inhibit the oxidative stress induced by SEA and protect the oxidative balance.It has been observed that PUN pretreatment can effectively improve mitochondrial damage and protect mitochondrial morphology in L02 hepatocytes.Meanwhile,the changes of mitochondrial autophagy-related protein content and the diminished occurrence of co-localization between autophagosomes and mitochondria all demonstrated that PUN pretreatment had a better alleviating effect on SEA-induced mitochondrial damage in L02 hepatocytes.In addition,compared to the SEA control group,the AST enzyme activity(U/L)in the high-dose PUN treatment group recovered from 56.28 to 37.27,and the ALT enzyme activity(U/L)decreased from 21.64 to 17.95,which gradually approached the level of the control group.At the same time,the content of apoptosis-related proteins also changed.These results demonstrated that PUN can protect mice from SEA induced liver injury.In summary,SEA can induce oxidative stress and mitochondrial autophagy by inducing the production of ROS,which leads to hepatocytes and liver injury,while PUN has a better protective effect on SEA-induced liver injury.