Mechanism Of Duodenal Ferroptosis In Rat Induced By Chronic Stress And The Protection Of Chlorogenic Acid

Chronic stress has a significant impact on the development and progression of animal diseases and can even be life-threatening.When chronic stress occurs,all organs are damaged and the intestine is one of the most vulnerable organs,while the duodenum is the main absorption site for iron,an essential element for life.When the duodenum is damaged by chronic stress,the body’s iron homeostasis is disrupted,causing various diseases and even inducing ferroptosis,which further leads to impaired nutrient absorption in the body caused by intestinal damage and seriously endangers the economic value of animals.It is important to identify the mechanisms involved in chronic stress-induced ferroptosis in the intestine and to search for appropriate protective drugs for veterinary clinical research.Chlorogenic acid has anti-inflammatory and antioxidant effects and it also has a protective effect on the intestine.Therefore,in this study we established a chronic stress model in rats and administered STAT3 inhibitors to explore the mechanism of duodenal ferroptosis caused by chronic stress and investigated the protective effect of chlorogenic acid on duodenal damage caused by chronic stress.In the experiment,56 healthy male Wistar rats were randomly divided into 7 groups: CON group,CS group,Cga+CON group,Cga+CS group,S3I-201+CON group,S3I-201+CS group and DMSO+CS group.Among them,CS group,Cga+CS group,S3I-201+CS group and DMSO+CS group were subjected to 6 h of fasciculation stress daily for 21 days,chlorogenic acid was given by gavage 1 h before moulding daily for Cga+CON group and Cga+CS group,and Corresponding drugs were given by intraperitoneal injection every 2 d for S3I-201+CON group,S3I-201+CS group and DMSO+CS group at 1 h before fasciculation.The rats were weighed every 3 days during model establishment,and the open field test was carried out at the end of model establishment.After testing,the rats were blood collected and executed and the tissues were frozen.The success of the chronic stress model in rats was determined by examining the changes in body weight,the results of the field test and the serum corticosterone level.The microstructural changes of duodenal tissues,the mRNA and protein expression of iron-regulated pathways in serum and liver,the mRNA and protein expression of indicators of regulation of intracellular iron intake and outflow in duodenum and the indicators of duodenal ferroptosis,and the iron content and distribution in tissues were used to clarify the mechanism of ferroptosis in duodenum caused by chronic stress and the protective effect of chlorogenic acid.The test results showed that the mean daily weight gain of the CS group was significantly lower than that of the CON group;the rats in the CS group were more anxious and had reduced exploratory abilities compared to the CON group;and the serum corticosterone levels were significantly higher in the CS group compared to the CON group(P<0.01).Histopathological results showed that the duodenal villi in the CS group were structurally disorganized;the duodenal damage in the S3I-201+CON group was alleviated compared to the CS group.The results of iron content and iron distribution staining showed that the expression of DMT1 protein and mRNA was unchanged in the CS group,while the expression of FPN1 protein and mRNA was significantly decreased in the CS group compared to the CON group(P<0.01).The results of ferroptosis-related indexes showed that compared with the CON group,the duodenal tissues of the CS group showed a significant increase in MDA and ROS(P<0.01),a significant decrease in GSH(P<0.01),a significant decrease in GPX4 protein expression(P<0.01),a significant increase in Ptgs2 mRNA expression(P<0.01)and a significant decrease in NADPH(P<0.01).The abnormal changes of MDA and ROS content,GSH content,GPX4 protein expression level,Ptgs2 mRNA expression level and NADPH content in the duodenal tissue of S3I-201+CON group were restored compared with the CS group(P<0.01).This indicates that chronic stress can cause an imbalance in duodenal iron homeostasis inducing ferroptosis and causing intestinal damage.The results of iron-regulator-related assays showed that serum iron-regulator content and liver iron-regulator mRNA levels were significantly increased in the CS group compared with the CON group(P<0.01);serum iron-regulator content and liver ironregulator mRNA levels were decreased in the S3I-201+CS group compared with the CS group(P<0.01).IL-6-related assays showed that serum IL-6 levels and liver IL-6 protein and mRNA levels were significantly higher in the CS group compared with the CON group(P<0.01).STAT3 pathwayrelated assays showed that liver IL-6,p-JAK2/JAK2 and p-STAT3/STAT3 protein expressions were significantly lower in the S3I-201+CS group compared to the CS group,restoring normal levels,suggesting that STAT3 is a key gene in hepatic iron-regulator synthesis and secretion and that chronic stress can cause duodenal ferroptosis through excessive activation of the STAT3-related pathway in the liver.After administration of chlorogenic acid,compared with the CS group,the duodenal tissue villi were relatively intact,the serum IL-6 and ferroregulin levels and the mRNA expression level of hepatic ferroregulin were significantly reduced(P<0.01),the protein and mRNA expression levels of duodenal DMT1 and FPN1 were significantly reduced(P<0.01),and the duodenal iron content was significantly reduced(P<0.01).Prussian blue staining did not show significant iron deposition,and the duodenal ROS and MDA levels were significantly reduced(P<0.01),GPX4 protein and mRNA expression levels were significantly increased(P<0.01),GSH levels were significantly increased(P<0.01),Ptgs2 mRNA expression levels were significantly reduced(P<0.01),and NADPH levels were significantly increased(P<0.01).This indicates that chlorogenic acid has a significant alleviating effect on chronic stress-induced duodenal ferroptosis.The results of these tests showed that chronic stress can increase the level of IL-6 in rats and overactivate the hepatic JAK2/STAT3 pathway,which can promote the synthesis of large amounts of iron-regulating hormone in the liver and cause the imbalance of iron homeostasis and iron accumulation in the duodenum,thus inducing the occurrence of intestinal ferroptosis.Chlorogenic acid can reduce the synthesis and secretion of hepcidin by inhibiting the hepatic IL-6/JAK2/STAT3 pathway,thereby alleviating chronic stress-induced duodenal ferroptosis in rats.