Protective Effect Of Berberine On Colon Injury In Cats By Regulating Intestinal Flora

Intestinal inflammation is one of the main pathological injuries that endanger the health of cats,and various factors can induce this damage.Berberine is a single component extracted from the traditional Chinese medicine Coptis chinensis,which has a good therapeutic effect on diarrhea and is widely used in the treatment of intestinal diseases in human medicine.Recent studies have shown that a variety of traditional Chinese medicines and their monomer components can protect intestinal tissue cells by regulating intestinal flora.This study in the cat colon inflammation as the main research object,analysis of berberine can effectively treat the cat intestinal inflammation,and analyze the regulation of intestinal flora,the intestinal inflammation injury and the contraction function regulation reveals the mechanism of berberine,expected for clinical cat intestinal inflammation disease treatment to develop new pet drugs.In this study,acute colon inflammation model was induced by sodium dextran sulfate（DSS）to simulate the clinical symptoms of uc.Twelve healthy adult shorthair cats half male and half female,body weight 2.0-2.3kg)were randomly divided into three groups:normal control group（CON）,DSS induced colitis group（DSS）and berberine treatment group（DSS+BBR）.The cat’s body weight and colon length were recorded.Metagenomic analysis technology was used to analyze the flora abundance and composition difference of the intestinal contents.The inflammatory response,cell death,intestinal tight junction and intestinal smooth muscle contraction related regulatory molecular genes and proteins were detected by histopathological analysis,real-time quantitative PCR and protein western blotting analysis.To verify the protective effect of berberine on colon inflammation and explore the role of intestinal flora in the treatment mechanism of berberine on colon inflammation.The results are as follows:（1）Clinical symptoms,disease activity index scores and colonic histopathological changesBody weight changes,colon length,disease activity index,and histopathological sections were examined in DSS induced feline colitis to assess the effect of BBR.The results showed that BBR had a protective effect on DSS induced colitis cats,significantly alleviating the symptoms of weight loss and colon shortening,and reducing the disease activity index.Pathological sections of colon H&E staining showed normal morphology of colon epithelial cells and no inflammatory cell infiltration in the control group,while a large number of inflammatory cell infiltration in the DSS group.In the BBR group,the inflammation was significantly reduced,and the cell structure of the whole section was similar to that of the control group.（2）Structural and functional changes of intestinal floraAfter the successful modeling of DSS,the composition of fecal microflora of cats changed greatly.BBR reshaped the intestinal microflora,Firmicutes increased,bacteroides decreased,and the proportion of both increased.The species accumulation curve showed that the amount of fecal samples collected was sufficient for species composition analysis in this experiment.By analyzing the species composition of each group,the experimental results showed that BBR led to the increase of prevotella,while DSS induced the decrease of proteus richness in colitis.Histogram of the relative abundance and Beta diversity analysis were used to compare the diversity of samples in each group.Both PCo A and PCA analysis showed that BBR cats had good intergroup repeatability,that is,they had higher similarity in fecal bacteria composition,while DSS showed a disorder in fecal bacteria composition.Cluster heat map analysis and random forest analysis of intestinal microbial communities showed that the balance between harmful and beneficial bacteria was restored after berberine treatment,and metabolic pathway diversity analysis showed that the abundance of metabolism-related pathways was the highest in the new city.The comprehensive results showed that BBR had a regulatory effect on intestinal flora and could maintain intestinal microbial homeostasis.（3）Analysis results of inflammatory signal molecules in colon tissueThe results of inflammatory genes showed that the expressions of Caspase-1,IL-1β,NLRP3,ASC,JAK2 and STAT3 in cat colon were significantly increased in DSS group（p<0.05）.The results show that BBR protects against colonic inflammation in cats by regulating the JAK2/STAT3 signaling pathway and activating the inflammasome NLRP 3,resulting in decreased expression of inflammatory factors（p<0.05）.（4）Analysis results of molecular analysis of cell death in colon tissueThe results showed that compared with CON group,the protein and m RNA expressions of Bax,Caspase-3,Caspase-7 and Caspase-12 genes in DSS group were increased,while the expression of Bcl-2 gene was decreased（p<0.05）.Meanwhile,p-Ml KL/MLKL in DSS group,The expression levels of p-RIP1/RIP1 and p-RIP3/RIP3 proteins were increased（p<0.05）,while BBR successfully inhibited the expression of the above genes,indicating that DSS could cause increased apoptosis of colon tissue cells and increased expression of programmed necrosis,that is,DSS caused increased death of colon tissue cells.BBR can mitigate these results.（5）Analysis results of colonic tight junction in colon tissueThe expression of m RNA and protein of N-Cadherin,E-Cadherin,Slug,ZO-1 and ZEB in DSS group were significantly decreased（p<0.05）.Compared with DSS group,m RNA and protein levels in DSS+BBR group were significantly increased.These results indicated that DSS affected the expression of colonic tight junction genes,and the tight junction of colonic epithelial cells was weakened.BBR could restore the damage of colonic tight junction induced by DSS to a certain extent.（6）Analysis results of the detection of smooth muscle related genes in colon tissueIt showed that Ca²⁺concentration in DSS group was significantly increased,the expression of Ca M was also increased,phosphorylated MLC expression was activated,smooth muscle contraction curve was strengthened,Ca M,MLCK,Rock,Rho A,m RNA and protein expressions were significantly increased（p<0.05）.After BBR treatment,these conditions were significantly relieved and gradually approached the normal level.This indicated that DSS caused colon smooth muscle injury,smooth muscle contraction phenomenon was obvious,BBR treatment after the colon smooth muscle contraction phenomenon was significantly relieved.These results indicated that DSS affected the expression of colonic tight junction genes,and the tight junction of colonic epithelial cells was weakened.BBR could restore the damage of colonic tight junction induced by DSS to a certain extent.In conclusion,this study confirmed that berberine can effectively protect colon tissue and relieve inflammatory damage in cats.Berberine can regulate the structure of intestinal flora,increase the proportion of beneficial bacteria and reduce harmful bacteria.Further regulate the JAK/STATs signal to reduce the level of inflammasome and inhibit inflammation;It also regulates key signaling molecules of apoptosis and programmed necrosis to prevent cell death and maintain the integrity of tissue structure.Regulate smooth muscle contraction and maintain the stability of colon tissue function in cats.Berberine plays a protective and therapeutic role on colon inflammation in cats by regulating intestinal flora in many aspects,which provides help for further application of berberine in the clinical treatment of pet intestinal diseases.