| Background and purpose Radiation-induced oral mucositis(RIOM)is one of the most common side effect in patients with head and neck undergoing radiotherapy,and 40-50% of them eventually develop into severe oral mucositis.Severe oral mucositis often leads to treatment interruption,prolonged patient’s treatment time,malnutrition,and increased hematological toxicity,resulting in reduced patient trearment efficacy.At present,there is no effective treatment method for the prevention of radiation-induced oral mucositis in the world.Therefore,there is an urgent need for effective drugs or methods to improve the oral mucosal inflammation caused by radiation.Thalidomide is a synthetic glutamic acid derivative.Recent studies have shown that thalidomide has a significant therapeutic effect on tissue inflammation.Small sample clinical studies have shown that thalidomide can improve oral mucosa related to disease treatment.However,the role of thalidomide in radiation induced oral mucositis still needs further clinical observation,and its regulatory mechanism needs to be further demonstrated.This study aim to investigate the protective effect of thalidomide on RIOM from the following two aspects:(1)To study the protective effect and regulatory mechanism of thalidomide on radiation oral mucositis through in vitro and animal experiments;(2)To explored the efficacy and safety of thalidomide in preventing oral mucositis in patients with nasopharyngeal carcinoma undergoing concurrent radiotherapy and chemotherapy.Methods(1)HOEC cell line(Human immortalized oral epithelial cell)was used for in vitro experiments.CCK8 assay and flow cytometry were used to detect cell apoptosis ratio to evaluate the effect of thalidomide on RIOM;Elisa and RT-q PCR experiments were used to assess the effect of thalidomide(THD)on the expression of inflammatory factors in HOEC cells after radiation;C57mice were used to build a RIOM model,and the effect of using or not using thalidomide on RIOM.(2)Using next-generation sequencing technology to analyze the effect of thalidomide on the expression profile of HOEC cells;using bioinformatics technology to analyze the cellular metabolic pathways and signal transduction pathways that thalidomide may be involved in.(3)RT-q PCR and Western blotting were used to assess the effect of thalidomide(THD)on the expression of downstream target molecules mi R-9-3p and NFATC2;Transient transfection technology was used to intervene the expression of mi R-9-3p and NFATC2 in HOEC cells.CCK8 assay,flow cytometry,dual luciferase assay,Elisa,and Western blot experiments demonstrated that the mi R-9-3p/NFATC2 regulatory axis inhibits the damage of oral epithelial cells under the radiation by regulating the NF-κB signaling pathway.(4)Multicenter,open label,randomized controlled trial was designed to explore the efficacy and safety of thalidomide in preventing oral mucositis in patients with nasopharyngeal carcinoma undergoing concurrent chemoradiotherapy from the clinical level.Results(1)Pre-treatment of HOEC cells with thalidomide(5μmol)could significantly reduce radiation-induced cell damage,which was manifested as enhanced cell proliferation,decreased apoptosis,and significantly decreased inflammatory factor concentrations in the culture system.The expression of pro-apoptotic proteins decreased,while the expression of anti-apoptotic proteins increased after treating HOEC cells with thalidomide;thalidomide can improve the degree of RIOM in mice,which showed that the epithelial layer of the tongue mucosa of mice in the thalidomide group remained relatively intact,complete,and continuous epithelial spikes and taste buds can be seen;at the same time,it was found that the infiltration of inflammatory cells and the proportion of apoptosis in the tongue mucosa of mice treated with thalidomide were significantly reduced.(2)Transcriptome sequencing results showed that compared with the irradiation group(RT),5230 genes were up-regulated and5,281 genes were down-regulated in cells treated with irradiation combine thalidomide(5μmol);Compared with irradiation alone(RT),2681 genes were up-regulated and 2550 genes were down-regulated cells treated with irradiation combine thalidomide(10μmol);the enrichment analysis based on differentially expressed genes showed that differentially expressed genes were involved in multiple cellular pathways and signal transduction pathways,especially inflammation and apoptosis-related pathways.(3)The results of in vitro experiments showed that thalidomide promoted the expression of mi R-9-3p in HOEC cells,while inhibited the expression of NFATC2;overexpression of mi R-9-3p could promote the proliferation of HOEC cells under irradiation,while reducing the apoptosis rate of HOEC cells;down-regulation of NFATC2 expression in HOEC cells can enhance the proliferation,inhibit the expression of inflammatory factors,and reduce the cell apoptosis ratio of cells under radiation;Conversely,up-regulation of NFATC2 in HOEC cells got the diametrically opposite result.Rescue experiments showed that overexpression of NFATC2 could attenuate the radiation protection effect of mi R-9-3p on HEOC cells and the inhibitory effect on NF-κB signaling pathway.(4)In the whole treatment period of nasopharyngeal carcinoma patients,the incidence of RIOM in the thalidomide group and the control group were 87.5% and 97.5%,respectively,and there was a statistically difference between the groups(p=0.016);the median time to onset of RIOM in the thalidomide group and control group was 30 days(interquartile range: 24-34 days)and 14 days(interquartile range: 12-17 days),respectively;Among the patients with RIOM,the proportion of patients with oral mucositis of grade 3 or above in the thalidomide group and the control group were 27.5% and 46.3%,respectively,and the comparison of the rates between the two groups was statistically significant(p=0.014);in terms of oral and pharyngeal pain,the proportion of patients with a pain score of 0 or 1 was higher in the thalidomide group than in the control group(23.75%,38.75% and 12.5%,35%,respectively),while the MTS score was lower in the thalidomide group than in the control group(20%,17.5% and 25%,27.5%,respectively);in terms of weight change,the amount of weight loss in the thalidomide-treated patients was significantly lower In the control group patients without thalidomide treatment(5.6±2.5kg and 4.3±1.7kg,respectively);in terms of short-term efficacy,the objective remission rates of the thalidomide-treated group and the control group were 96.3%(77/80)and93.8%(75/80),there was no statistically significant difference between the two groups;Among the adverse events in the two groups,the incidence of constipation and nausea in the thalidomide group was higher than that in the control group,while the rates of vomiting,dizziness and insomnia were lower than those in the control group.The rates and types of remaining adverse events were similar in the two groups.Conclusion(1)Thalidomide can inhibit radiation-induced oral epithelial cell injury,including inhibiting radiation-induced apoptosis,inflammatory factors and pro-apoptotic gene expression,and promoting the expression of anti-apoptotic protein Bcl-2;Pre-intervention of mice with thalidomide can reduce the degree of radiation-induced oral mucosal inflammatory response in mice.(2)Thalidomide regulates radiation-induced oral epithelial cell injury involving multiple cellular pathways and signaling pathways,especially pathways related to inflammation and apoptosis.Thalidomide can also regulate alternative splicing of oral epithelial cells.(3)Thalidomide inhibits the activation of NFATC2-mediated NF-κB signaling pathway by promoting the expression of mi R-9-3p,thereby exerting a radioprotective effect on oral mucosa.(4)In patients receiving concurrent chemoradiotherapy for NPC,thalidomide can prolong the incubation period of oral mucosal inflammation and reduce the incidence of oral mucosal inflammation,with acceptable safety,and does not affect the short-term efficacy. |
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