Puerarin mediated miR-30b-5p targeting fibroblast activation protein against oral submucous fibrosis

Background:Puerarin(Pue)has been reported to be a natural active ingredient with multiple antifibrotic properties.This work aimed at exploring the function of Pue in oral submucousfibrosis(OSF)treatment.Methods:Human oral mucosafibroblasts(hOMF)were induced with transforming growth factor beta1(TGF-β1)and intervened with Pue.Expressions offibrosis-related markers were analyzed by Western blot and IF staining.Cell viability was characterized by the CCK-8 assay.Expressions of miR-30 family members were quantified by qRT-PCR.The correlation betweenfibroblast activation protein(FAP)and miR-30 family expression was evaluated by the Pearson correlation coefficient.Bioinformatics prediction and dual-luciferase reporter assay were employed to verify the regulation between FAP and miR-30b-5p.The specific mechanism of Pue on OSF was explored through the promotion or inhibition of miR-30b-5p.Results:After induction by TGF-β1,hOMF showed upregulated Collagen I,Collagen III,and FAP expressions,while miR-30 family expression was downregulated with miR-30b-5p being the most significant.Pue intervention inhibited the excessive proliferation of TGF-β1-induced hOMF,downregulated FAP,collagen type 3(COL3A1),collagen type 1(COL1A1),matrix metalloproteinase 1(MMP1),and matrix metalloproteinase 3(MMP3)expressions,and restored miR-30 family expression.Bioinformatics prediction and dual-luciferase reporter assay revealed that miR-30b-5p selectively inhibited FAP expression.Mechanistically,miR-30b-5p mimic suppressed the excessive proliferation of TGF-β1-induced hOMF and declinedfibrosis levels.Pue intervention significantly reversed the promotion of TGF-β1-induced OSF by miR-30b-5p inhibition.Conclusion:Pue mediated miR-30b-5p targeting FAP against OSF,which provided a theoretical basis for the pathogenesis research and Pue application in OSF.

Pre-assembled nanospheres in mucoadhesive microneedle patch for sustained release of triamcinolone in the treatment of oral submucous fibrosis

Objective:Drug-loaded mucoadhesive silk fibroin(SF)microneedle patch can overcome the limitations of low bioavailability and significant pain associated with traditional treatment methods,such as topical application or injection of triamcinolone for oral submucous fibrosis(OSF).However,these systems release the drug too quickly,failing to meet the clinical requirements.This study aims to construct a mucoadhesive SF microneedle patch pre-assembled with silk fibroin nanospheres(SFN)and explore its ability to sustain the release of triamcinolone in the treatment of OSF.Methods:SFN was pre-assembled via precipitation reaction and characterized by scanning electron microscope(SEM)for the morphology.The particle size andζ-potential were measured by dynamic light scattering(DLS).Triamcinolone was loaded onto SFN using a diffusional post-loading method.The effective loading of triamcinolone was confirmed using Fourier-transform infrared spectroscopy(FTIR).The concentration of unloaded triamcinolone was quantified by high-performance liquid chromatography.Drug encapsulation efficiency and loading capacity of SFN were then calculated to determine the optimal amount of drug loading.The SFN suspension was pre-mixed with SF solution to prepare the microneedle under-layer.The microneedle morphology was observed by SEM.Compression mechanical tests were performed to evaluate the fracture force of microneedles at different nanosphere contents(5%,10%,and 20%),determining the optimal pre-mixing ratio.Ex-vivo mouse oral mucosa permeation studies were performed to ascertain the insertion depth of the microneedles via histological sections.The adhesive top layer was synthesized using SF and tannic acid,with FTIR confirming its successful synthesis.Its viscoelasticity was characterized by a rheometer,and differential scanning calorimetry analyzed thermal properties.Tensile tests evaluated the interfacial bonding strength between the adhesive layer and microneedle base to ensure no detachment during use.Adhesion to wet oral mucosal tissues was tested and compared to commercial oral patches.Under the optimized conditions,the double-layered mucoadhesive microneedle patch with pre-assembled nanospheres was prepared.Its cell compatibility was evaluated by cell counting kit-8(CCK-8),live/dead staining,and phalloidin staining after co culturing with fibroblasts.The drug release experiment was conducted to demonstrate its sustained release efficacy.Results:SFN(mean diameter 46.25 nm)was successfully prepared.The maximum drug encapsulation efficiency was(63.88±1.09)%(corresponding loading capacity of SFN was(27.41±3.06)%when the weight ratio of triamcinolone/SFN was 0.5.The corporation of SFN did not affect microneedle morphology.The mechanical properties of microneedles decreased with increasing nanosphere amount.Only the fracture force of the group with 5%SFN[(0.07±0.01)N/needle]exceeded the minimum force required for mucosal penetration,thus selected as the optimal pre-mixing ratio.Histological sections confirmed that the SFN microneedles could penetrate the epithelial layer and deliver drugs to OSF affected areas.Adhesion strength between the microneedle base and top layer was(94.8±6.89)kPa,confirming strong bonding with no detachment during use.The wet adhesive strength of the double-layered mucoadhesive microneedle patch[(41.28±7.43)kPa]was significantly enhanced compared to commercial oral patches(4.5 kPa,P<0.01).CCK-8 and live/dead staining results confirmed no significant cytotoxicity.Drug release experiment showed the double-layered mucoadhesive microneedle patch with pre-assembled SFN enabled sustained release time of triamcinolone from 4 days to 14 days.Conclusion:Pre-assembling nanospheres in mucoadhesive SF microneedle patches can extend triamcinolone release time,meeting clinical requirements for sustained drug delivery.

MORPHOMETRIC ANALYSIS OF SPINOUS CELL IN ORAL SUBMUCOUS FIBROSIS COMPARISON WITH NORMAL MUCOSA, LEUKOPLAKIA AND SQUAMOUS CELL CARCINOMA

The morphometric analysis of the spinous cell in 16 specimens of oral submucous fibrosis (OSF) was made by using interactive image analysis system (IBAS-II). 19 parameters of the size and shape were chosen, and compared with normal mucosa, leukoplakia, dysplasia and carcinoma. The results indicated that the cell dimensions (area, perimeter, all kinds of diameter) and nuclear cytoplasmic ratio in OSF were between normal mucosa and dysplasia as well as carcinoma. The former showed a progressive decrease (P<0.01), and the latter showed a progressive increase (P<0.01). The dimensions of the nuclei did not show considerable differences among the groups (P>0.05). A series of discriminant functions had been developed with stepwise discriminant analysis, the agreement ratio for OSF was 93.75%. The decrease of cell area and the increase of nuclear cytoplasmic ratio could reflect a malignant progress. The cell morphometric model could discriminate OSF well from other groups, suggesting that the change of the epith

Development of a mouse model of arecoline-induced oral mucosal fibrosis

Objective: To develop a BALB/c mouse model of oral submucous fibrosis(OSF)induced by arecoline and to exhibit an accumulation of collagen and angiogenesis changes.Methods: BALB/c mice were randomly assigned to either the control(distilled water) or experimental group(arecoline)(n = 40). Eight mice from each group were sacrificed every 4 weeks since 8 weeks post treatment. Changes in histopathologic features, levels of collagen type Ⅰ and collagen type Ⅲ, and angiogenesis were measured.Results: In the 8th week, epithelium atrophy, collagen cumulation and micrangium pathologic changes in the lamina propria were observed in the oral mucosa. In the 20th week, hyaline degeneration of the connective tissues was observed on the tongue and palate mucosa. The angiogenesis and collagen type Ⅰ changed significantly as the diseases advanced(P < 0.05); however, collagen type Ⅲ was not statistically different.Conclusions: An OSF model involving mice can be rapidly induced by drinking a highdose of arecoline. OSF angiogenic changes in mice primarily decrease and collagen accumulation is mainly collagen type Ⅰ.

Advances in molecular mechanisms of oral submucosal fibrogenic carcinogenesis

Oral submucosal fibrosis(OSF)is a chronic,progressive and insidious oral mucosal disease.Its development is highly associated with areca chewing and eventually evolves into oral squamous cell carcinoma(OSCC),which is currently recognized as a precancerous state by the International Center for Research on Cancer(IARC).Due to the extremely complex carcinogenesis process of OSF,the carcinogenesis mechanism is not clear,and most scholars believe that the disease is the result of multiple factors.Therefore,this paper mainly summarized the latest research on the molecular mechanism of OSF canceration,aiming at understanding the law of OSF canceration and providing theoretical reference for early detection,diagnosis and treatment of OSF canceration.

Effect of Huoxue Huayu Detoxification on GSK-3β and β-catenin levels in OSF patients and its mechanism

Objective:To explore the effects of Huoxue Huayu detoxification method on the levels of GSK-3βandβ-catenin in OSF patients and its mechanism.Methods:A total of 120 patients with oral submucosal fibrosis(OSF)treated in our hospital from May 2018 to December 2019 were selected and divided into routine treatment group and Huoxue Huayu detoxification group of 60 cases according to the lottery method.The routine treatment group was treated with triamcinolone acetonide injection,and the Huoxue Huayu detoxification group was treated with Huoxue Huayu detoxification.The lesion area,mouth opening degree,SFRP1 level in gingival crevicular fluid and saliva,TCM symptom score,TGF-β1,IL-6,GSK-3β,β-catenin expression and clinical effect were observed in the two groups.Results:After treatment,the lesion area of oral mucosa in the Huoxue Huayu detoxification group was smaller than that in the routine treatment group(P<0.05),and the mouth opening degree in the Huoxue Huayu detoxification group was larger than that in the routine treatment group(P<0.05);After treatment,the level of SFRP1 in gingival crevicular fluid and saliva and clinical effect in the Huoxue Huayu detoxification group were higher than those in the routine treatment group(P<0.05);the TCM syndrome score,TGF-β1,IL-6,GSK-3β,β-catenin expression in the Huoxue Huayu detoxification group were lower than those in the routine treatment group(P<0.05).Conclusion:By acting on Wnt/β-catenin signaling pathway,Huoxue Huayu detoxification method can regulate the expression of TGF-β1,IL-6,GSK-3β,β-catenin,improve the clinical symptoms of patients with oral submucous fibrosis,alleviate the body inflammation,alleviate the pain,with remarkable effect.

Collagen membrane for reconstruction of soft tissue defects after surgery of oral cancer and precancer:a brief review

There are some surgeries after which a temporary cover for raw wounds is required to ensure healing.Some of those circumstances are loss of tissue due to burns,trauma,amputation,chronic ulcer,leprosy,and skin graft sites.Although the body initiates regeneration mechanisms,however the time taken for complete healing of wounds is unpredictable.Also,there is a tendency for long standing wounds to undergo infection and scarring.Oral mucosa is no exception to scarring and infection of wounds and there has always been a search for new materials that can be used for coverage of oral defects.Xenogenous collagen is one such grafting material.Over the years collagen implant solutions for a number of clinical applications include general surgery,burn surgery,neurosurgery,plastic and reconstructive surgery,oral surgery,and peripheral nerve and tendon surgery.This paper aims to focus on collagen as an effective option of wound closure in plastic and reconstructive surgery of the head and neck,especially after loss of soft tissue following resection of oral malignancies.