| As a major subtype of inflammatory bowel disease(IBD),Ulcerative colitis(UC)can cause diarrhea,abdominal pain,rectal bleeding,weight loss and other symptoms.UC increases the risk of life-threatening colon cancer if the patient’s disease continues to deteriorate or is not treated effectively for a long time.Although the exact mechanism of the disease is still unclear,studies have shown that genetic diversity and environmental factors are closely related to the development of IBD.That is to say,IBD is the result of the combination of the above factors.At present,the anti-ulcerative colitis drugs mainly include anti-inflammatory drugs,immunomodulators,corticosteroids,biological agents,etc.,but long-term use will cause users to produce diarrhea,indigestion and other side reactions,and it is arduous to meet the needs of efficient and safe modern clinical treatment.With the organic integration of nanotechnology in various directions,especially for the biomedical field,nanodrugs equipped with many advantages can alleviate or even overcome some limitations of conventional drugs and are gradually incorporated into the clinical treatment of diverse diseases.The research scope of liposome nanovesicles referring to a variety of natural plants has been expanded in recent years.To our relief,scientists have extracted diverse lipid vesicles from edible plants,which contain many naturally active ingredients linked to the effective treatment of some diseases.It can be said that plant lipid nanovesicles with the characteristics of wide source,low cost and high efficacy have certain application potential in clinical practice.At present,pharmacological studies have found that Dendrobium officinale Kimura et Migo,a perennial herb of Dendrobium Orchidaceae,has antioxidant,anti-inflammatory,anti-tumor,anti-fatigue effects and has the ability to protect gastric mucosa and so on.In this study,a large number of Dendrobium officinale lipid nanovesicles(DONPs)were extracted and isolated from stems of Dendrobium officinale as the research object to explore its therapeutic effect on ulcerative colitis in mice.To be specific,the research content of this paper covers the following parts:The first part is focusing on the characterization analysis and cell experiment of DONPs.In this study,DONPs were obtained from Dendrobium officinale by differential centrifugation and gradient centrifugation.At the characterization level,the particle size and surface potential of DONPs were analyzed by dynamic light scattering particle size analyzer,and the morphology of DONPs was observed by atomic force microscope and transmission electron microscope.Next,the lipid component analysis and stability evaluation of the nanomedicine were carried out.As for the in vitro cell experiment of DONPs,MTT assay was used to detect the toxicity of DONPs to macrophage Raw 264.7.Flow cytometry(FCM)and laser confocal scanning microscopy(CLSM)were utilized to quantitatively and qualitatively detect the cellular uptake efficiency.The antioxidant activity of DONPs was evaluated comprehensive by DPPH assay and the measurement of the changes on intracellular ROS reactive oxygen cluster content.The expression level of TNF-αin macrophage Raw 264.7was detected by ELISA kit after DONPs treatment.Conclusions:The shape of DONPs is spherical,the particle size is about 193nm,the surface potential is-6.54 m V,and the DONPs is rich in lipid composition.The stability of DONPs in gastrointestinal simulated fluid is relatively good with relatively stable particle size.In vitro cell experiments showed that Dendrobium officinale derived lipid nanovesicles had good biocompatibility for Raw 264.7 macrophages.DONPs,to some extent,can inhibit the he over-secretion of TNF-αin LPS-induced macrophages in a certain concentration range.Meanwhile,DONPs can inhibit oxidative stress of Raw 264.7 macrophages induced by LPS.Macrophages phagocytosis of DONPs is time-dependent,and through quantitative phagocytosis experiments,we found that DONPs can promote the phagocytosis of macrophages to a certain extent,verifying its natural galactose targeting.The second part focuses on the therapeutic effect of DONPs on UC mice in vivo.Firstly,in order to measure the absorption of the drug in the colon and five viscera of mice,in vitro imaging technology was used to trace certain amount of Di O fluorescent dye labeled DONPs in UC mice at different time points.Through the intake of different protein concentrations of DONPs,combined with a number of macro and micro parameters of UC,the therapeutic effect of oral DONPs on UC in vivo was tested.Conclusions: Animal experiments have confirmed that oral administration of DONPs can make these natural nanovesicles accumulate in the colon.Besides,we found that it is 12 hours that ranked first for the accumulation amount of DONPs in the colon.Oral administration of DONPs inhibited macroscopic symptoms such as weight loss and colon shortening in UC mice afterwards.In the meantime,DONPs can down-regulate the expression of TNF-α in serum of UC mice and reduce MPO activity and other microscopic parameters,highlighting its effective relieving effect on colitis.The third part is concerned with the evaluation of lipid nanovesicles derived from Dendrobium officinale on intestinal microorganism.Taking the flora in the feces of mice in the UC in vivo treatment experiment as the experimental research object,the corresponding situation of intestinal microbes of different groups of mice was analyzed from many aspects,such as the richness and uniformity of the composition of the bacterial colony,the changes of the composition of the bacterial colony at the phylum level and so on.Conclusions: The results of diversity,composition and richness of flora indicate that lipid nanovesicles from Dendrobium candidum have the ability to maintain the stability of intestinal microflora,and play a therapeutic role on UC by regulating intestinal microflora and restoring its diversity.The fourth part involves evaluation of DONPs in vivo.In line with the administration frequency and maximum dose level of UC treatment in mice,DONPs were orally administered to healthy mice for several time.The safety of DONPs in vivo was comprehensively evaluated from the aspects of body weight,blood routine,hepatorenal toxicity,tissue staining and so on.Conclusions: There is no significant difference in the macro and micro parameters of all mice receiving oral DONPs compared with the blank control group.The results indicated that lipid nanovesicles derived from Dendrobium candidum have good biological safety.As mentioned above,Dendrobium officinale derived lipid nanovesicles have good biocompatibility,anti-inflammatory,and antioxidant properties.This project not only opens a new road for the innovative application of Dendrobium candidum,provides a theoretical basis for the development and utilization of new resources,but also enriches the research system for the treatment of colitis with natural edible plant derived lipid nanovesicles. |
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