Anti-Inflammatory Effects Of Rhein Loaded Nanomicelles In Periodontitis Bone Loss&Cases Report

Inflammatory bone diseases that occur in the maxillofacial region,such as periodontitis and periapical periodontitis,can affect the patient’s chewing function,and in severe cases,can cause tooth loosening and ultimately tooth extraction.At present,there are no therapeutic drugs for inflammatory bone diseases,and the treatment of periodontitis and periapical periodontitis mostly relies on mechanical therapy assisted with drug therapy.For teeth with deep periodontal pockets or refractory periapical periodontitis,adjuvant drug therapy can improve the curative effect to a certain extent.Oral administration has the problem of short duration of time and low bioavailability.Therefore,it is necessary to develop a new anti-inflammatory medicine with good safety and sustained release ability for local use to alleviate maxillofacial bone inflammation.As a natural antioxidant,rhein has a wide range of pharmacological properties,such as anti-inflammatory,antioxidant,antibacterial and anti-osteoclast abilities.Rhein is a potential therapeutic agent for inflammatory bone diseases.However,its poor water solubility and stability hinder its clinical application.Liposome is a widely used drug delivery carrier,which can increase the solubility and stability of encapsulated drugs and has good biocompatibility.In this study,liposome was used as a carrier to prepare rhein loaded nanomicelles to improve the physical and chemical properties of rhein.The anti-inflammatory effects of rhein loaded nanomicelles in maxillofacial bone inflammation were investigated both in vitro and in vivo,providing a new idea for the clinical treatment of maxillofacial inflammatory bone diseases.Part 1: Preparation and characterization of the rhein loaded nanomicellesPurposes: To prepare the rhein loaded nanomicelles(Rh-NMs)and investigate their physicochemical properties such as particle size,Zeta potential,water-solubility,stability,and release rate in vitro.Methods: The Rh-NMs were prepared by self-assembly of distearoylphosphatidylethanolamine-N-poly(ethyleneglycol)2000(DSPE-PEG)through a facile nano precipitation method.A fluorescence spectrometer was used to investigate the fluorescence intensity of rhein and the Rh-NMs,and the absorption spectra was measured by a UV-vis spectrophotometer.The encapsulation efficiency and uploading efficiency of rhein in DSPE-PEG 2000 were measured by the UV-visible spectrophotometer.The morphology of Rh-NMs was observed under a transmission electron microscope.Zetasizer Nano analyzer was used to investigate the particle size,polydispersity index and Zeta potential of the Rh-NMs.The dissolution of rhein solution and Rh-NMs solution was observed at room temperature.Then the release rate of rhein in Rh-NMs was studied by dialysis method.Results: The UV-visible spectrum and fluorescence spectrum of Rh-NMs showed the same representative peaks as rhein,indicating that rhein was successfully encapsulated in Rh-NMs.Under the transmission electron microscopy,Rh-NMs were spherical with a diameter of about 260 nm.The results from the Zetasizer Nano analyzer showed that the average particle size of Rh-NMs was 274.90 ± 9.75 nm,the Zeta potential was-16 ± 0.42 m V,the encapsulation efficiency was 76.62%,and the drug loading efficiency was 13.28%.In addition,the particle size and Zeta potential of Rh-NMs were measured for 7 consecutive days without significant changes,indicating the stability of Rh-NMs.After standing at room temperature for 24 hours,the Rh-NMs solution was homogeneous and stable,while precipitation could be seen in the rhein solution,which confirmed the water solubility and stability of Rh-NMs.In Rh-NMs,rhein was released continuously for the first 24 hours,and then the drug release rate slowed down.Within 72 hours,a cumulative of 70% of the drug was released,indicating that the release of rhein in Rh-NMs was a continuous process.Conclusions: In this study,we prepared rhein-loaded nanomicelles which showed satisfactory water solubility,stability and sustained release capability.Part 2: Anti-inflammatory and antioxidant ability of the Rh-NMsPurposes: To investigate the cytotoxicity of rhein and Rh-NMs,their effects on cell uptake,and their anti-inflammatory and antioxidant effects in cell model of inflammatory response.Methods: We investigated the cytotoxicity of rhein and Rh-NMs on RAW 264.7 cells and human gingival fibroblasts through Cell Counting Kit-8 and Calcein/PI Live/Dead Viability/Cytotoxicity Assay Kit analysis.The cell uptake of rhein and Rh-NMs was observed under microscope.0.5 μg/m L lipopolysaccharide(LPS)was used to induce inflammation in RAW 264.7 cells.A Reactive Oxygen Species Assay Kit was used to detect the antioxidant effect of the rhein and Rh-NMs.Real-time quantitative PCR and Western blotting were used to detect the effect of rhein and Rh-NMs on inflammatory cytokines and the potential mechanisms.Results: The results of cell proliferation assay showed that rhein and Rh-NMs at ≤ 20μM had no effect on the proliferation of RAW264.7 cells and human gingival fibroblasts.The results of live/dead staining showed that there was no significant change in cell morphology and no increase in the proportion of dead cells after treatment with rhein and Rh-NMs for 24 hours.The results of cell uptake test showed that the drug entered macrophages and played a role over time.Under the induction of 0.5 μg/m L LPS,RAW264.7 cells produced a large amount of reactive oxygen species,and the level of inflammatory factors in the cells increased,indicating that the cell model of inflammatory response was successfully constructed.After pretreatment with rhein and Rh-NMs,the expression of inflammatory factors such as IL-1β,IL-6 and TNF-α decreased,and the expression of phosphorylated NF-κB P65 and phosphorylated IKB-α decreased.In addition,in RAW264.7 cells pretreated with rhein and Rh-NMs,LPS-induced ROS production was reduced,the expression of Nuclear factor-erythroid 2-related factor 2(Nrf2)in the nucleus was increased,and the expression of Heme Oxygenase-1(HO-1)was significantly increased.Conclusions: Rhein and Rh-NMs had good compatibility on cells at experimental concentrations and inhibited the activation of NF-κB pathway and the expression of inflammatory factors as well as the production of ROS in LPS induced cells.The antioxidant effect may be related to the promotion of Nrf2 into the nucleus to increase the expression of HO-1.Part 3: Protective effect of Rh-NMs in experimental periodontitis in micePurposes: To investigate the sustained-release ability,biocompatibility and the protective effect of Rh-NMs for local use in mice with periodontitis.Methods: DiR labeled Rh-NMs solution and free DiR solution with the same concentration was injected into the palatal gingiva of mice and the local fluorescence intensity was acquired immediately after injection and 2,6,12,24 hours after intervention to evaluate degree of drug metabolism.The mouse periodontitis model was constructed by ligation of the maxillary second molar with silk thread.PBS,rhein or Rh-NMs of the same concentration were injected into the palatal gingiva of the maxillary second molar.At day 9,the mice were euthanized,the maxillary bone was collected,and the alveolar bone height and bone tissue volume was observed and measured by stereo microscope and Micro-CT.The maxillary tissue was collected,decalcified,embedded in paraffin and sliced.Hematoxylin-eosin(HE),tartrate resistant acid phosphatase(TRAP)staining,Masson staining and immunofluorescence analysis were used to evaluate the morphology of alveolar bone and the degree of local tissue inflammation.To study the biocompatibility of Rh-NMs,the blood of the mice was collected for blood routine analysis.After euthanasia,the main organs of the mice were collected and stained with HE to evaluate histological changes under the microscope.Results: Free DiR solution in the gingiva decreased rapidly after 2 hours,and it was almost completely metabolized after 12 hours.In contrast,DiR labeled Rh-NMs was sustained released,and the local fluorescence intensity was 78% of the initial fluorescence intensity after 24 hours.The results of stereomicroscope and Micro-CT showed that the alveolar bone of mice with silk ligation and local injection of PBS was significantly absorbed,the alveolar bone height and the bone volume was reduced.Local injection of rhein or Rh-NMs could reduce the alveolar bone absorption induced by ligation to varying degrees.The results of HE staining showed that the integrity of periodontal tissue in the ligation + PBS group and the ligation + rhein group was destroyed,and the alveolar bone was absorbed to varying degrees.In Ligation + RhNMs group,periodontal tissue was relatively intact and the alveolar bone absorption was mild.TRAP staining results showed that local injection of rhein or Rh-NMs could reduce the number of osteoclasts in alveolar bone.Masson staining showed that the periodontal tissue fibers in the Ligation + PBS group were sparsely arranged while the collagen fibers in Ligation + Rh-NMs group were denser and more ordered.In addition,we detected the expression of TNF-α in gingival tissues by immunofluorescence staining,and the results showed that more inflammatory cytokines were expressed in the Ligature + PBS group and the expression of TNF-α in the Ligature + Rh-NMs group was the lowest.The results of biocompatibility experiment showed that the blood routine of mice in each group was within the normal level and there was no significant difference between mice in each group.Also,no pathological changes were observed in HE staining of visceral tissue.Conclusions: Rh-NMs had sustained release ability for local use in the gingival of mice,and the sustained release time was more than 24 hours.In periodontitis model of mice,local injection of rhein or Rh-NMs could reduce periodontal tissue inflammation and alveolar bone absorption to varying degrees.Moreover,rhein and Rh-NMs had good biocompatibility for local use.