The Effects And Mechanism Of Mulberry Leaf Flavonoids On Ulcerative Colitis

Mulberry（Morus alba）,a deciduous plant with strong adaptability,wide distribution,and rapid growth,contains one of the most valuable and rich natural components found in mulberry plants.China,as the largest planting country in the world,has a long history of planting mulberry trees and has an abundant supply of mulberry leaves.Due to their high protein content,mulberry leaves are an important resource for livestock feed,and the polyphenolic compounds（mainly flavonoids）found in mulberry leaves offer good anti-inflammatory and antioxidant properties,with beneficial effects on animal health and production.Mulberry leaves,as traditional Chinese medicine,have a variety of biological activities and are widely used to treat various diseases.Ulcerative colitis（UC）is a chronic non-specific inflammatory bowel disease,with typical clinical symptoms consisting of abdominal pain,diarrhea,and bloody stools,which can seriously affect the daily life of patients.Currently,drugs used to treat UC mainly include 5-aminosalicylic acid,corticosteroids,and immunosuppressants.These drugs have certain therapeutic effects,but their long-term use will weaken patient immunity,and they have a high cost.Therefore,it is very important to screen for low-cost,low-toxicity,and effective candidate therapies from natural plant-derived bioactive substances.Flavonoids offer good anti-inflammatory activity,and mulberry leaves are rich in flavonoids.In this study,we selected Mulberry leaf flavonoids（MLFs）with the best anti-inflammatory activities from MLFs eluted by different ethanol concentrations（30%,50%,and 75%）,and toxicological tests were used to determine the safe concentration range,to further evaluate the pharmacological properties of the MLF with the best anti-inflammatory and antioxidative activity in dextran sulfate sodium（DSS）-induced UC in mice.The main investigations and results were as follows.1.Anti-inflammatory and antioxidant activities of MLFsMulberry leaf flavonoids（MLFs）were eluted with 30%,50%,and 75%ethanol to obtain 30%,50%,and 75%MLF,respectively,of which the 30%MLF had the highest total flavonoids content.A nontargeted metabolomic analysis technique was used to analyze 24types of differential flavonoids between the MLFs.Quercetin,kaempferol,and their derivatives were found to be more abundant in 30%MLF than in the other two MLFs.In addition,their anti-inflammatory and antioxidant activities were compared to the inflammation model of RAW 264.7 mouse cells induced by lipopolysaccharide（LPS）.The results showed that three types of MLFs inhibited the production of nitric oxide（NO）,prostaglandin E2（PGE2）,inducible nitric oxide synthase（i NOS）,cyclooxygenase-2（COX-2）,and inflammatory cytokines in the LPS-induced RAW 264.7 cells.All MLFs enhanced the antioxidative capacity by decreasing reactive oxygen species（ROS）production and the scavenging of 2,2-diphenyl-1-picrylhydrazyl（DPPH）free radicals,as well as by improving the metal ion chelating activity and reducing power.Furthermore,30%MLF showed a better anti-inflammatory and anti-oxidant effect than 50%MLF and75%MLF.2.Toxicity study of 30%MLFAcute toxicity and subacute toxicity tests were conducted to explore the safe concentration range of 30%MLF.An acute toxicity study was performed at a single dose of20 g/kg for 14 consecutive days,while a subacute toxicity test was conducted via the daily oral administration of 30%MLF at doses of 2.5,5,and 10 g/kg for 28 days.The acute toxicity study showed that the LD₅₀ of 30%MLF exceeded 5 g/kg,while the subacute toxicity results showed no significant adverse effects of 30%MLF at 2.5,5,and 10 g/kg.No toxicity was observed in the liver,spleen,and kidneys in the mice,as determined by hematological,serum biochemical,and histological analyses via the daily oral administration of 30%MLF at 2.5,5,and 10 g/kg.The above results indicated that30%MLF had a large safe dosage range.3.Alleviating the effect and mechanism of 30%MLF on DSS-induced UC in miceThe anti-inflammatory properties of 30%MLF were explored by using a DSS-induced mouse UC model.The results showed that 30%MLF treatment could mitigate the symptoms of DSS-induced colitis in mice,such as the loss of body weight,decreased food intake,diarrhea,bloody stools,colon shortening,and spleen hypertrophy.In addition,DSS induction could cause typical pathological characteristics of colitis,including the infiltration of inflammatory cells,damaged glands,the loss of crypts,and colonic mucosa erosion.Under DSS administration accompanied by 30%MLF treatment,the colon tissues exhibited relatively intact intestinal mucosa and glands,low infiltration of inflammatory cells,and minimal crypt damage,with low histological scores.In addition,30%MLF had a protective effect on the intestinal barrier of the UC mice.In the UC mice treated with 30%MLF,the decreased number of goblet cells,as well as the loss of mucin MUC2 and tight junction proteins（claudin-1 and ZO-1）,was significantly alleviated.The 30%MLF could also reduce the expression of inflammatory factors（TNF-α,IL-1β,and IL-6）in DSS-induced mice colitis to alleviate the inflammatory response.Immunohistochemistry（IHC）and Western blot（WB）analysis were used to explore the mechanism by which 30%MLF relieves UC in mice.The IHC results showed that 30%MLF could inhibit the protein expression of TLR4 and My D88,and the WB results showed that30%MLF could significantly inhibit the phosphorylation of key proteins in the MAPK pathway（ERK1/2,p38,and JNK）and NF-κB pathway（p65 and IκBα）activated by DSS.The 30%MLF also significantly inhibited the activation of the TLR4/My D88 pathway and the phosphorylation of key proteins in MAPK and NF-κB.Bay11-7085,an inhibitor of NF-κB,was used to treat the LPS-induced RAW 264.7 cells,and we found that Bay11-7085significantly inhibited the production of NO,PGE2,and the inflammatory cytokines（TNF-α,IL-1β,and IL-6）.In conclusion,30%MLF could alleviate DSS-induced UC in mice,and the involved pathways included the TLR4/My D88/MAPK/NF-κB pathway.4.Effect of 30%MLF on intestinal flora and metabolic pathway of DSS-induced UC in miceThe 16S r DNA amplicon sequencing results showed that after 30%MLF treatment,the flora structure of the UC mice was similar to that of the control group.The 30%MLF could increased the relative abundance of certain probiotics（such as Blautia,Lachnoclastridium,Lachnospiraceae＿NK4A136＿group,and Ligilactobacillus）and the content of acetic acid and butyric acid in the intestinal contents of the UC mice.In addition,metabolomics technology was used to detect and analyze the metabolites of the intestinal contents of the mice with UC after treatment with 30%MLF.The results showed that30%MLF could alleviate the metabolic disorders of amino acids and their metabolites and glycerol phospholipid metabolites in UC.The main metabolic pathways involved were those related to phospholipid metabolism,alanine,aspartate,and glutamate metabolism,and D-glutamine and D-glutamate metabolismIn conclusion,the 30%MLF had a good anti-inflammatory effect,and it potentially mitigated DSS-induced UC in mice by decreasing inflammatory factors,protecting intestinal barrier functionality,and ameliorating intestinal flora,as well as regulating alanine,aspartate,and glutamate metabolism and phospholipid metabolism.The TLR4/My D88/MAPK/NF-κB pathway was potentially involved in the anti-inflammatory mechanism of 30%MLF.Therefore,this study may be of great significance to the study of the anti-inflammatory activity and mechanism of 30%MLF,offering valuable information for the diversified development and utilization of mulberry leaves as a functional food and non-antibiotic feed in animal husbandry.