The Intervention Of Luteolin On Non-alcoholic Fatyty Liver Disease And The Preparation Of Colon-targeted Nanoparticles

Objective: Non-alcoholic fatty liver disease（NAFLD）has been shown to be closely associated with disturbances of the intestinal microbiota and intestinal mucosal barrier.Luteolin is a natural flavonoid with anti-inflammatory,antioxidant and anti-tumor activities.The purpose of this study was to investigate whether luteolin alleviates NAFLD induced by high fat diet（HFD）and the possible mechanisms involved in the gut-liver axis.According to the mechanism of action,luteolin colon-targeted nanoparticles（Lu-NPs）were prepared and characterized in vitro to meet the requirements of colon-targeted drug delivery.Methods: NAFLD model of rat was established by feeding high fat diet（HFD）,and luteolin was given intragastric administration.The serum levels of total cholesterol（TC）,triglyceride（TG）,high density lipoprotein cholesterol（HDL-C）,low density lipoprotein cholesterol（LDL-C）,alanine aminotransferase（ALT）and aspartate aminotransferase（AST）in rats were detected by kit to evaluate the status of blood lipid and serum transaminase levels.Fasting insulin（FINS）and fasting blood glucose（FPG）levels were detected with the kit,and HOMA-IR index was calculated.The liver was stained with eosin and hematoxylin（HE）to observe the degree of pathological damage.The levels of TG and TC in liver of rats were detected by kit,and the lipid accumulation in liver were determined by oil red O staining.The contents of lipopolysaccharide（LPS）,interleukin-6（IL-6）,interleukin-1 β（IL-1β）and tumor necrosis factor α（TNF-α）in serum of rats were determined by kit.The expression of TLR-4 and NF-κB m RNA in rat liver was detected by real-time quantitative PCR.Western blotting was used to detect the protein expression of nuclear NF-κB p65 and phosphorylated NF-κB P65（P-NF-κB P65）in liver tissues,and to evaluate the effect of luteolin on TLR-4inflammatory pathway involved in NAFLD.The permeability of rat intestinal mucosa was measured by valgus method.The m RNA expressions of tight junction proteins claudin-1,occludin and ZO-1 in rat intestine were detected by real-time quantitative PCR.The protein expression of tight junction proteins claudin-1,occludin and ZO-1 in intestinal tissue was detected by western blot.HE staining and immunohistochemical staining were performed to evaluate the intervention effect of luteolin on intestinal mucosal barrier injury in NAFLD rats.16 S r RNA gene sequencing was used to analyze the diversity of intestinal flora and the classification of intestinal flora at different classification levels,so as to evaluate the recovery of luteolin on the dysbiosis of NAFLD rats.Lu-NPs was prepared by emulsion solvent evaporation method,and the preparation conditions were optimized by response surface methodology.Transmission electron microscopy（TEM）was used to observe the appearance of the nanoparticles.The particle size,zeta potential and polydispersion index（PDI）were measured by malvin laser particle size analyzer.The drug loading and encapsulation rate of nanoparticles were determined,and the release and stability of nanoparticles in vitro were studied.Results: The weight and liver index of NAFLD rats were decreased after luteolin intervention.Serum TC,TG,LDL-C,AST and ALT were significantly decreased,HDL-C was significantly increased,and blood lipid and transaminase were improved.Serum FINS and FPG decreased significantly,and HOMA-IR decreased,suggesting that luteolin improved insulin resistance.The levels of liver TG and TC decreased significantly.HE staining showed that luteolin significantly improved liver injury.Oil red O staining showed that luteolin reduced lipid accumulation in liver.LPS was decreased in serum of rats.The levels of TLR-4,NF-κB,IL-6,IL-1β and TNF-α in liver were significantly decreased,indicating that inflammation in liver was reduced.Ameliorated the increased permeability of intestinal mucosa to fluorescein isothiocyanate-dextran（FD-4）induced by high-fat diet.The expression of claudin-1,occludin and ZO-1 in intestinal tissue increased significantly,and the permeability of intestinal mucosa decreased.HE staining results showed that the intestinal mucosal injury was relieved.Sequencing results of 16 S r RNA gene showed that luteolin reversed the proportion of Bacteroidetes and Firmicutes at the phylum level,and changed the difference of intestinal bacterial composition at the family level,thus increasing the diversity of intestinal flora in NAFLD rats and alleviating intestinal flora disorder.Lu-NPs were successfully prepared with spherical appearance,average particle size of197.45±20.09 nm,PDI of 0.22±0.01,average zeta potential of-23.5±1.16 m V,drug loading of 5.67±0.25%,encapsulation rate of 76.41±8.24%.After entering the colon environment（simulated with PBS solution of p H 7.4）,the cumulative release amount reached 88.27±9.33% within 4 h,which was consistent with the basic characteristics of oral colon-targeted nanoparticles and had good stability within 4weeks.Conclusions: Luteolin has a good therapeutic effect on HFD-induced NAFLD,inhibits liver inflammation,reduces intestinal mucosal barrier permeability and restores intestinal flora ecological environment through gut-liver axis.The prepared luteolin colon-targeted nanoparticles located the release site of the drug in the colon,so as to achieve better therapeutic effect locally and provide new ideas and methods for the treatment of NAFLD.