Therapeutic Effect Of Pterostilbene Nanoparticles On Ulcerative Colitis And Its Mechanism

Background:Ulcerative Colitis（UC）is a non-specific inflammatory bowel disease whose etiology and pathogenesis have not been fully elucidated,affecting more than 3.5 million people worldwide.The prevalence of UC has increased dramatically over the past decade and is a major public health problem worldwide.Studies have shown that the imbalance of intestinal ecological balance is an important risk factor for UC,which is mainly manifested as intestinal immune disorder and elevated oxidative stress level.In recent years,natural small molecule compounds have attracted much attention in the field of UC therapy due to their advantages of safety,low toxicity and various biological activities.Pterostilbene（PTE）is a kind of natural small molecule compound derived from blueberry and other plants.It has anti-inflammatory,antioxidant and other biological functions,showing a high therapeutic potential.However,due to the poor water solubility and low bioavailability of PTE,the applicability of PTE has not been paid much attention.With the continuous development of nanotechnology,more and more nanoparticles have been developed and used to improve the bioavailability and water solubility of natural small molecule compounds,thereby improving their therapeutic effects.In this study,polyethylene glycol（PEG）was conjugated to PTE to form amphiphilic polymers that self-assembled into nanoparticles PTENPs in water to improve the water solubility and bioavailability of PTE.Objective:Preparation of pterostilbene nanoparticles（PTENPs）in order to improve the bioavailability of PTE,to improve its solubility in water,thus improve its therapeutic effect on UC,explore its potential mechanism.Methods:PEG-PTE was prepared by esterification of Methoxy polyethylene glycol carboxylic acid(m PEG_2k-COOH)and PTE.The PEG-PTE was characterized by Fourier infrared spectroscopy（FT-IR）,ultraviolet spectrophotometer（UV/VIS）and nuclear magnetic resonance hydrogen spectroscopy（¹H-NMR）.PTENPs were self-assembled by thin film dispersion method,and characterized by Zeta potential and nanoparticle size analyzer and transmission electron microscope（TEM）.The stability of PTENPs was evaluated by Zeta potential and nanoparticle size analyzer.The release of PTENPs in buffer solution with esterase was studied.The uptake of PTENPs by RAW264.7 cells was observed by confocal laser microscopy.The biosafety of PTENPs was evaluated by measuring in vitro cell level and in vivo tissue level.Lipopolysaccharide（LPS）was used to stimulate RAW264.7 to construct an in vitro cell inflammation model.Flow cytometry（FCM）,Western blot（WB）,enzyme-linked immunosorbent assay（ELISA）,immunofluorescence（IF）and other related experimental techniques were used.The anti-inflammatory and antioxidant activities and potential mechanisms of PTENPs were investigated in vitro.The acute ulcerative colitis model of C57BL/6J mice was established by 3%dextran sulfate sodium（DSS）.The colonic distribution of PTENPs was observed by small animal imaging（IVIS）.Disease activity index（DAI）,H&E staining,body weight change and colon length were used to evaluate the therapeutic effect of mice.The expression of inflammatory signaling pathway（NF-κB/MAPK）in colon tissue was detected by WB,and the secretion of inflammatory factors in colon tissue and serum was detected by ELISA.The expression of transcription levels of inflammatory factors in colon tissues was detected by q RT-PCR,the polarization-related proteins of macrophages in colon tissues were detected by immunohistochemistry（IHC）,the subtypes of mouse spleen macrophages were detected by FCM,and the protein expression of tight junction（TJP-1）in colon tissues was detected by IF.The contents of malondialdehyde（MDA）and glutathione（GSH）were detected,and apoptosis of colon tissue sites was detected by d UTP in situ incision end labeling（TUNEL）mediated by terminal deoxynucleotide transferase.Results:PEG-PTE was successfully synthesized,and its drug loading was 11.2%.PTENPs prepared by thin film dispersion method showed spherical shape,and the hydration nanoparticle size was 124.6 nm,showing good stability.Free PTE can be released under the action of esterase.Compared with free PTE,PTENPs can reduce its cytotoxicity（P<0.05）and can be effectively taken up by RAW264.7 cells.Injecting 100mg/kg/d of PTENPs into the tail vein does not affect the body weight of mice,nor does it lead to the change of tissue morphology of the major organs of mice.In vitro cell experiments showed that PTENPs inhibited the activation of Toll-like receptor 4（TLR4）and the downstream NF-κB/MAPK signaling pathway.The secretion and expression of interleukin-6（IL-6）,interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）and nitric oxide（NO）were inhibited,and the polarization of RAW264.7 cells to M1 type was inhibited（p<0.05）.By scavenging excessive reactive oxygen species（ROS）,inhibiting malondialdehyde（MDA）content and increasing glutathione（GSH）content in lipid peroxidation（p<0.05）,it could protect cells from ROS-induced damage and inhibit cell apoptosis.In vivo animal experiments showed that compared with healthy mice,tail vein injection of PTENPs could accumulate more in the inflammatory colon tissue site of UC mice through ERP effect（p<0.05）.PTENPs could regulate the TLR4-mediated NF-κB/MAPK signaling pathway,regulate the secretion and expression of inflammatory cytokines,and inhibit macrophage polarization to M1 type（p<0.05）.Compared with the control group,the DSS-induced acute ulcerative colitis was ameliorated by inhibiting the MDA content in the lipid peroxidation reaction and increasing the GSH content（p<0.05）,inhibiting the apoptosis of colon tissue,up-regulating the expression of intestinal tight junction protein（TJP-1）,and repairing intestinal epithelial barrier.Conclusion:PTENPs have been successfully prepared,which has good stability and biocompatibility.It can passively target to the inflammatory tissue site of colon,and release free PTE under the action of esterase,and then play its biological functions.PTENPs can inhibit the activation of TLR4,inhibit the activation of NF-κB/MAPK signaling pathway,regulate the polarization of macrophages,reduce the release of inflammatory factors,and exert its anti-inflammatory effect.PTENPs can reduce oxidative stress,protect cells from cell damage caused by ROS,maintain the integrity of intestinal epithelial cells,and alleviate acute ulcerative colitis in mice through the synergistic effect of exerting its antioxidant capacity and enhancing the activity of antioxidant enzymes in vivo.Significance:This study can provide theoretical basis for using nanotechnology to improve the bioavailability of natural small molecule compounds and improve the efficacy of UC,which has very important scientific significance and clinical application value.