Effects Of Alkaloids From Mulberry Leaves On D-Galactose-Induced Oxidative Injuration In Mice And Its Underlying Mechanism

A large number of studies have confirmed that oxidative stress in the intestine can induce a variety of inflammatory intestinal diseases and intestinal flora disorders,and affect the digestion and absorption function of the intestine.This article firstly explored the effects of mulberry leaf alkaloids on D-galactose(D-Gal)-induced intestinal oxidative damage in mice,and discussed its protective effects on the intestinal barrier and its mechanism.This experiment was performed by injecting 1000 mg/kg·BW D-galactose(D-Gal)intraperitoneally for 45 days to establish a intestinal oxidative damage model.After the model was successfully established,the low-dose,medium-dose and high-dose groups were intragastrically given 50,100 and 200 mg/kg·BW mulberry leaf alkaloids,respectively,the positive control group was given 200 mg/kg·BW glutathione(GSH),the normal group and model group were given normal saline,for 45 days.During the experiment,the growth of the mice and the body weight were recorded.After the experiment,the mice were sacrificed,blooded,small intestine and other samples were collected.The pathological observation of the small intestine was carried out by HE staining,and related indexes were determined by ELISA kit and RT-PCR.The main conclusions were as follow:(1)In this study,mulberry leaf alkaloids can effectively improve the intestinal oxidative stress damage in mice caused by D-Gal.(1)Mulberry leaf alkaloids can effectively improve the oxidative damage of biological macromolecular in the small intestine.Compared with the model group,the lipid oxidation markers 4-HNE and 8-iso-PG2α in the high-dose group were reduced by 42.58% and 53.13%,respectively(P<0.05).The protein oxidation markers PCO and Dityr were reduced by 38.28%,41.12%,respectively(P<0.05),protein sulfhydryl groups increased by 62.44%(P<0.05).DNA oxidation markers 8-OH-d G,γ-H2 AX were reduced by 53.58%,50.61%,respectively(P<0.05).The effects of high-dose mulberry leaf alkaloids on the above indicators were equivalent to that of GSH(P>0.05),but only PCO,Dityr,protein sulfhydryl,8-OH-d G returned to the normal levels.(2)Mulberry leaf alkaloids can effectively remove ROS,and enhance the antioxidant capacity of the intestine.Compared with the model group,the levels of ROS in the high-dose group decreased by 30.34%(P<0.05),and the activities of antioxidant enzymes SOD,GST,NQO1 and HO-1 increased by 63.59%,68.61%,38.63% and 73.13%,respectively(P<0.05).The levels of non-enzymatic antioxidant substances GSH and Trx-1 increased by 29.19% and 123.75%,respectively(P<0.05).The effects of high-dose mulberry leaf alkaloids on the above indicators were equivalent to that of GSH(P>0.05),but only SOD,GST,HO-1,and GSH returned to the normal levels.(2)In this study,mulberry leaf alkaloids had protective effects on intestinal barrier of model group mice.(1)Mulberry leaf alkaloids can effectively repair the structural damage of intestinal physical barrier in model mice and improve its integrity and permeability.Compared with the model group,the levels of D-LA,DAO,LPS,and i FABP in the serum of mice in the high-dose group were reduced by 22.14%,23.22%,32.12%,and19.98%,respectively(P<0.05).The effects of high-dose mulberry leaf alkaloids on the above indicators were equivalent to that of GSH(P>0.05),but only D-LA,DAO,and LPS returned to the normal levels.The results of HE staining showed that mulberry leaf alkaloids can reduce the structural damage of small intestine,restore the tightly arranged structure of intestinal epithelial cells.(2)Mulberry leaf alkaloids can significantly improve spleen and thymus index,and alleviate the inflammatory response in small intestine,effectively improve the damage of intestinal immune barrier in model mice.Compared with the model group,the levels of IL-1β and IL-12β in the high-dose group were reduced by 35.88% and32.92%(P<0.05),the levels of TGF-β and IL-10 were increased by 76.06%,101.31%,respectively(P<0.05).The effects of high-dose mulberry leaf alkaloids on the above indicators were equivalent to that of GSH,but only IL-1β and TGF-β returned to the normal levels.(3)Mulberry leaf alkaloids can improve the intestinal flora structure disorder in model mice and protect the intestinal biological barrier.High-throughput sequencing technology analysis results showed that high-dose mulberry leaf alkaloids can significantly improve the richness and diversity of intestinal flora,adjust the ratio of Firmicutes/Bacteroides,and reduce the abundance of potentially harmful bacteria such as Lachnospiraceae＿NK4A136＿group,Helicobacter,and Mucispirillum,increase the abundance of potential probiotics such as Lactobacillus and Bifidobacterium,and returned to normal levels.(3)In this study,mulberry leaf alkaloids have good regulatory effects on the gene expression of key factors in the Keap1/Nrf2 antioxidant pathway,tight junction related proteins and TLR4/My D88/NF-κB inflammation pathway in the small intestine.(1)Compared with the model group,Keap1 m RNA expression in the high-dose group decreased by 297.77%(P<0.05),and the expression of Nrf2,NQO1,and HO-1m RNA increased by 111.88%,124.37%,125.37%,respectively(P<0.05).The effects of high-dose mulberry leaf alkaloids on the above indicators were equivalent to that of GSH,but only Keap1,NQO1,and HO-1 m RNA returned to the normal levels.Correlation analysis showed that Nrf2 m RNA expression was highly positively correlated with the activity of antioxidant enzyme and the contents of non-enzymatic antioxidant.(2)Compared with the model group,the expression of ZO-1,Occludin,and Claudin-1 m RNA in the high-dose group increased by 154.67%,272.78%,and 81.16%,respectively(P<0.05).The effects of high-dose mulberry leaf alkaloids on the above indicators were equivalent to that of GSH,but only Occludin and Claudin-1 m RNA returned to the normal levels.Correlation analysis showed that the expression of ZO-1,Occludin,and Claudin-1 m RNA was moderate negatively correlated with the levels of D-LA,DAO,LPS,and i FABP.(3)Compared with the model group,the expression of TLR4,My D88,and NF-κB m RNA in the high-dose group were reduced by 50.08%,63.10%,and 60.54%,respectively(P<0.05).The effects of high-dose mulberry leaf alkaloids on the above indicators were equivalent to that of GSH,but only the expression of TLR4 m RNA returned to the normal levels.Correlation analysis showed that the expression of TLR4,My D88,NF-κB m RNA was highly positively correlated with IL-1β and IL-12β,but was highly negatively correlated with TGF-β and IL-10.In summary,mulberry leaf alkaloids can significantly improve D-Gal-induced small intestinal oxidative stress damage in mice.Its mechanism of action includes:(1)By activating the Keap1-Nrf2/ARE antioxidant signal pathway,improve small intestinal oxidative damage.(2)By regulating the expression of tight junction protein ZO-1,Occludin,and Claudin-1 m RNA,improve the integrity and permeability of the intestinal physical barrier.(3)By inhibiting the expression of TLR4/My D88/NF-κB m RNA,alleviate inflammation and improve the intestinal immune barrier damage.(4)By restoring the diversity and richness of the intestinal flora and regulating the structure of the flora,protect the intestinal biological barrier.