Protective Effect Of Tanshinone ⅡA On Vomiting Toxin Induced Damage To Porcine Small Intestinal Epithelial Cells

Mycotoxins are very common in clinical production and life.The production of molds in cereal raw materials and feedstuffs is affected by various factors such as temperature,humidity,and storage methods.Contaminated feedstuffs contain a large amount of mycotoxins,which have adverse effects on animal bodies.Animals infected with mycotoxins often experience clinical symptoms such as weight loss,decreased immunity,reproductive disorders,and mental distress.Vomitoxin(DON)is a relatively common type of mycotoxins,which is widely concerned due to its wide distribution,strong toxicity,and synergistic effects with other toxins.Pigs are the most sensitive animals to DON.When DON enters animals,it can produce the same symptoms as most mycotoxins such as aflatoxin,zearalenone toxin,ochratoxin,etc.,such as refusal to eat,weight loss,mental depression,diarrhea,and can damage various organs and systems in the body through blood circulation.Currently,antibiotics and physical methods are commonly used to prevent and treat DON,which can lead to problems such as animal resistance and unabsorbability of nutrients.As a natural plant,Chinese herbal medicine has the advantages of green safety,small intestinal irritation,small liver and kidney damage,and no drug residue.Therefore,there is an urgent need to develop relevant traditional Chinese medicine preparations,conduct in-depth research on the effective ingredients and pharmacological properties of traditional Chinese medicine,and produce alternatives that are more consistent with animal food safety and environmental and ecological safety.Tanshinone IIA is an active substance extracted from traditional Chinese medicinal material Salvia miltiorrhiza.Modern pharmacological studies have shown that tanshinone IIA has multiple biological activities such as anti-inflammatory,antioxidant,antiviral,anti-tumor,and also has a certain role in maintaining intestinal homeostasis.This experiment took porcine small intestinal epithelial cells(IPEC-j2)as the research object,and used the DON induced IPEC-j2 cell injury model as the experimental platform to explore the effect of tanshinone ⅡA on alleviating IPEC-j2 cell injury,providing experimental and theoretical basis for the prevention and treatment of clinical DON toxin,and also provide certain medication reference for animal poisoning caused by clinical DON.The test is divided into two parts:Part I: DON-induced damage to IPEC-j2 cellsDigestive system damage caused by DON can harm animal health,and even lead to animal death.This is related to oxidative stress,inflammation,and apoptosis of intestinal cells caused by DON.Therefore,it is very necessary to determine the occurrence and severity of damage to intestinal cells caused by DON.(1)Selection of DON concentrationIn this study,to investigate the effect of DON on the damage of IPEC-j2 cells,we first treated IPEC-j2 cells with different concentrations of DON(0.01 μ M,0.05 μ M,0.1 μ M,0.2 μ M,0.5 μ M,1 μ M,2 μ M,5 μ M,10 μ M)for 24 hours,and then detected the cell viability using the CCK-8 method.The results showed that when the concentration of DON was 0.5 μ M,it had a significant impact on the viability of IPEC-j2 cells(P<0.001);When the concentration of DON is 2 μ M,the cell viability is greater than 50%.Therefore,DONs with concentrations of 0 μ M,0.5 μ M,1 μ M,and 2 μ M were selected as the conditions for the cell damage model to be treated for 24 h.(2)Effects of DON on oxidative stress,inflammation,and apoptosis in IPEC-j2 cellsAfter treating IPEC-j2 cells with different concentrations of DON(0 μ M,0.5 μ M,1 μM,2 μ M)for 24 hours,the effects of different concentrations of DON on the expression levels of lactate dehydrogenase(LDH),malondialdehyde(MDA),and reactive oxygen species(ROS)in IPEC-j2 cells were detected.The results showed that with the increase of DON concentration,the intracellular LDH,MDA,and ROS levels significantly increased(P< 0.001).Subsequently,real-time fluorescence quantitative method was used to detect the expression levels of oxidative stress,inflammation,and apoptosis related genes such as Mn-SOD,Cu Zn-SOD,IL-6,Bax,and Caspase-3.The results showed that with the increasing concentration of DON,the intracellular oxidative stress level,inflammation level,and apoptosis level were significantly increased(P < 0.001).Western blot results also showed that with the increase of DON concentration,the intracellular inflammatory related protein IL-1 β,the expression levels of P65 and P-P65 in nuclear protein were significantly increased(P < 0.001).The above results indicate that DON can cause damage to IPEC-j2 cells,and the damage gradually increases with the increase of DON concentration.(3)Effects of DON induced IPEC-j2 cell damage on intercellular tight junctionsAfter treating IPEC-j2 cells with different concentrations of DON for 24 hours,the effect of DON induced IPEC-j2 cell damage on cell tight junction was investigated.The results showed that treating IPEC-j2 cells with different concentrations of DON significantly inhibited the expression of tight junction related genes such as Occludin,N-cad,ZO-1,Claudin-1,and Claudin-3.Western blot analysis also showed that DON significantly inhibited the expression of tight junction related proteins such as Occludin,N-cad,ZO-1,Claudin-1,and Claudin-3 in IPEC-j2 cells.This indicates that DON destroys the tight connection between IPEC-j2 cells.(4)Effect of DON on mitochondrial autophagy in IPEC-j2 cellsAfter treating IPEC-j2 cells with different concentrations of DON for 24 hours,the effects of DON induced damage on the mitochondria of IPEC-j2 cells were investigated.The results showed that treating IPEC-j2 cells with different concentrations of DON significantly increased the expression level of mitochondrial autophagy related genes such as LC3,P62,PINK1,and Parkin.Western blot detection of related proteins showed the same trend,indicating that DON induced the occurrence of mitochondrial autophagy in IPEC-j2 cells.In order to further detect the degree of mitochondrial damage,the mitochondrial membrane potential(JC-1)and mitochondrial permeability transition pore(m PTP)were detected and it was found that DON significantly decreased the mitochondrial membrane potential of IPEC-j2 cells and caused the mitochondrial permeability transition pore to open abnormally.This indicates that DON causes mitochondrial damage in IPEC-j2 cells.Part II: Tanshinone Ⅱ A alleviates DON induced IPEC-j2 cell damageThe first part of the test results showed that DON can cause damage to IPEC-j2 cells.In order to alleviate this injury,this experiment selected one of the three extracts of Chinese herbal medicine,namely,yam polysaccharide,chlorogenic acid,and tanshinone IIA,to explore its alleviation effect on the damage to IPEC-j2 cells caused by DON.(1)Selection of Effective Components and Concentrations of Chinese Herbal MedicineFirst,the use concentration of three effective ingredients of Chinese herbal medicine was selected through CCK-8 test,and then the concentration was selected as 45 through CCK-8 test μ G/m L of tanshinone Ⅱ A was co cultured with IPEC-j2 cells as a therapeutic concentration.(2)Effects of tanshinone Ⅱ A on expression levels of oxidative stress,inflammation,and apoptosis in DON-induced IPEC-j2 cellsCell morphology observation test found that tanshinone IIA effectively alleviated adverse effects such as abnormal cell morphology and reduced density.After detecting the expression levels of LDH,MDA,and ROS,it was found that tanshinone Ⅱ A significantly decreased its expression level(P < 0.001).After that,the expression of genes and proteins related to oxidative stress,inflammation,and apoptosis was detected again,and it was found that tanshinone Ⅱ A significantly reduced the level of intracellular oxidative stress,inflammation,and apoptosis(P < 0.001)The above results indicate that tanshinone Ⅱ A can reduce the levels of oxidative stress,inflammation,and apoptosis in IPEC-j2 cells.(3)Mitigation effect of tanshinone Ⅱ A on damage of tight junction between IPEC-j2 cells induced by DONThis experiment was conducted to investigate the protective effect of tanshinone Ⅱ A on DON induced damage to IPEC-j2 cells,and to detect cell tight junctions.The experimental results showed that tanshinone Ⅱ A significantly increased(P < 0.001)the expression of tight junction related genes and proteins,and alleviated cellular tight junction damage caused by DON.(4)Mitigation effect of tanshinone Ⅱ A on mitochondrial damage in IPEC-j2 cells induced by DONIn order to further explore the role of tanshinone Ⅱ A in alleviating DON induced damage to IPEC-j2 cells,this experiment conducted relevant tests on cell mitochondria.The results showed that after the addition of tanshinone IIA,the expression of mitochondrial autophagy related genes and proteins LC3,P62,PINK1,and Parkin were significantly reduced compared to the DON group(P < 0.001),and tanshinone IIA significantly improved the problems of decreased mitochondrial membrane potential and abnormal opening of mitochondrial permeability transition pores.This indicates that tanshinone Ⅱ A effectively alleviates mitochondrial damage in IPEC-j2 cells caused by DON through the PINK1-Parkin pathway.The results of this experiment indicate that DON can cause oxidative stress in IPEC-j2 cells,increase the expression levels of LDH,MDA,and ROS in cells,and cause intracellular inflammation,apoptosis,and mitochondrial autophagy,resulting in damage to intercellular tight junctions,leading to cell damage.Tanshinone Ⅱ A can alleviate the damage to IPEC-j2 cells caused by DON by reducing the expression levels of LDH,MDA,and ROS,reducing the expression of genes and proteins related to oxidative stress,inflammation,apoptosis,and mitochondrial autophagy,and increasing the expression of tight junction related genes and proteins.The data and conclusions of this trial provide a new medication reference for the clinical prevention and treatment of vomiting toxins,and provide a corresponding theoretical basis for the development of new drugs for the prevention and treatment of vomiting toxins.