The Role And Mechanisms Of Diospyros Spp. Extracts In Rat Model With Experimental Monophasic And Relapsing ROU

Oral ulcers (recurrent oral ulceration, ROU) is the most common oral mucosal disease with the prevalence rate of 20%, as one of the most common complications in AIDS, Behcet’s disease, or cancers. There have been no ideal drugs and strategy for the treatment of ROU so far. With emerging many problems along with traditional antibiotics or corticosteroids used for ROU, such as no good effect, many side effects, etc., it is undoubtedly necessary and good choice for traditional Chinese medicine and natural medicine to treat ROU. Although there were several clinical cases for persimmon fruit or leaves to treat oral ulcers in ancestral prescription books in China, and also clinical reports in recent years, there is lack of experimental evidence about the effect for persimmon fruit or leaves on ROU.In this study, two effective components from Niuxin persimmon and data plum persimmon for ROU were collected initially by several bioseparation methods such as EtOH/NH4 (SO4)2 aqueous two-phase system, enzymatic deproteinization and ultrafiltration membranes. By autoantigen injected SD rat model with relapsing ROU had been established. Pathogenesis of relapsing ROU and the effect for total polysaccharides from persimmon fruit on relapsing ROU were discussed from the perspective of CD4+ T cell subsets containing Th17, Treg and related cytokines. Furthermore the possible signaling pathways of TPsPF had been searched in LPS-induced mouse macrophage. Besides, the role of TPDPPL was investigated in monophasic ROU after injected SD rat with 1% HCL.The main conclusions are as follows:1. After TPDPPL obtained from data plum persimmon leaves, the EtOH/(NH4) 2SO4 aqueous two-phase systems were used for TPDPPL refining and purification. Response surface methodology (RSM) was utilized as a statistical tool and optimized experimental conditions with highest rate of TPDPPL were found to be EtOH mass fraction 5.0% and (NH4)2SO4 mass fraction 20.0% at pH value 7.0. Meanwhile after the enzymatic deproteinization method was optimized of TPsPF obtained from Niuxin persimmon fruit, TPsPF of 10-200 kDa were enriched using two molecular weight cut-off(MWCO)membrane (200 kDa,10 kDa)and the optimal condition were selected. The results showed average retention rate of TPsPF of 10-200 kDa at 45.82%(RSD 2.37%).2. A rat model with experimental relapsing ROU were established by autoantigen injected with only CFA adjuvant injected as the normal control group. Histopathological slice showed oral mucosa had partially dissolved and disappeared in MODEL group. ELIS A results showed that Treg-related cytokines concentrations of TGF-β1 and IL-10 were significantly decreased in the serum, and Th17-related cytokines levels of IL-6 and IL-17 were significantly up regulated in MODEL group. On the other hand, FCM analysis showed the proportion of CD4+ CD25+ Foxp3+ Treg cells had a very significant reduction in MODEL group. At last, RT-PCR had been used for analyzing the transcription factors Foxp3, RORyt mRNA expression in tissue and the results showed Foxp3 mRNA expression significantly reduced, while RORyt mRNA expression significantly increased in MODEL group. These results indicated that the differentiation of CD4+ T cells in ROU model have shifted to Th17 cells from the perspective of Th17 and Treg cells subsets. Dynamic changes of TGF-β1, IL-17 in rat peripheral blood with relapsing ROU indicated IL-17 level have very significant differences before ulceration, after ulceration, compared with during ulceration. While TGF-β1 level have differences before ulceration, during ulceration, compared with after ulceration. The conclusion provide a reasonable explanation for self-healing and a clue to explore further the reason for recurrence in ROU.3. After rat model with relapsing ROU treated with three different dosage of TPsPF and Prednisone Acetate, the effects were evaluated refer to clinical therapeutic effect criterion of ROU. High TPsPF have significantly extended the intervals, reduced the number of ulcers, and significantly reduced the ulcer area, with denoted I1N1 and evaluated as markedly effective. ELIS A result showed that high TPsPF can significantly enhance TGF-β1 level which promote self-healing and significantly reduce levels of IL-6 and IL-17 in serum. FCM detected the proportion of CD4+ CD25+ Foxp3+ Treg cells have greatly increased by 1.5 times in high TPsPF group compared with model group. The result of transcription factors Foxp3 mRNA, RORyt mRNA expression in tissue showed middle TPsPF and high TPsPF could significantly increase the expression of Foxp3 mRNA and TPsPF could significantly reduce the expression of RORyt mRNA. We guess high TPsPF maybe play a role in ROU by promoting CD4+ CD25+ Foxp3+ Treg differentiation and inhibiting Th17 differentiation.4. RAW264.7 mouse macrophage cells had been cultured in vitro. TPsPF in a dose-dependent manner significantly inhibited NO production, inhibited IL-6 levels, down-regulated TLR4 mRNA expression, significantly suppressed IκBa protein degradation and NF-κB p65 protein phosphorylation. TPsPF may inhibit pro-inflammatory gene activation via TLR4/NF-κB signaling pathway and further suppressed the secretion of pro-inflammatory cytokines for example IL-6.5. High TPDPPL could reduce ulcer area and significantly down-regulate IFN-y level, while middle and high TPDPPL could significantly shorten ulcer duration in rat model with monophasic ROU. Meanwhile middle TPDPPL and high TPDPPL could inhibit acute ear swelling induced by xylene in KM mice. High TPDPPL may play an important role in monophasic ROU model by reducing IFN-y levels and inhibit acute inflammation. Analgesic test indicated high TPDPPL could increase the threshold of the pain by tenderness in mice within 30 min, while the acute analgesic effect was not obvious.