| Research purposes:Radiation esophagitis(RE)is a common adverse reaction of radiotherapy for cervical and thoracic tumors.It is a serious complication that limits treatment and affects the quality of life.There is a lack of effective treatment.Studies have shown that radiation esophagitis injury is mainly divided into two parts:acute inflammatory injury and fibrosis repair.The transcription of inflammatory factors mediated by the NF-κB pathway is the core pathological mechanism of the inflammatory injury period,and fibrosis and mucosal repair are the fibrosis repair period.main pathological changes.The traditional Chinese medicine Kuiyangye prepared based on the theory of external treatment of traditional Chinese medicine can be absorbed through the mucous membrane,and has achieved significant clinical curative effect in the treatment of radiation mucositis,but the curative effect on radiation esophagitis is not significant,which still needs to be improved.In recent years,the construction of nano-hydrotalcite drug-loading system provides new research ideas for improving the efficacy of traditional Chinese medicine.This study is divided into two parts:clinical and basic:In the clinical research,an open-label randomized controlled research method was selected to compare the therapeutic effects of traditional Chinese medicine Ulcer Liquid and the commonly used clinical drug Kangfuxin Liquid on the treatment of radiation esophagitis;in the basic research,a rat model of radiation esophagitis was constructed,to verify the superiority of nano-Kuiyangye compared with traditional dosage forms and positive drugs,to explore the mechanism of nano-Kuiyangye to regulate inflammation,promote repair,and inhibit pain,and to clarify the mucosal adhesion and tissue absorption of nano-hydrotalcite carrier for traditional Chinese medicine ulcer fluid rate increase.Research methods:1 Clinical research:Using the method of open-label randomized controlled clinical research,49 patients with radiation esophagitis were randomly divided into positive control group and treatment group.The changes of radiation esophagitis grade(CTC-2.0 version),pain score(NRS score),nutritional status(weight,albumin,prealbumin,hemoglobin)and treatment drug onset time were compared between the two groups before and after treatment.,to evaluate the clinical efficacy of traditional Chinese medicine Ulcer Liquid compared with Kangfuxin Liquid in the treatment of radiation-induced esophagitis.The drug safety of ulcer liquid was evaluated by comparing the changes of liver function and kidney function and the occurrence of adverse reactions in the two groups of patients before and after treatment.2 Basic research:2.1 Pharmacodynamic evaluation of nano-Kuiyangye in treating radiation esophagitis in rats:Construct and optimize the rat model of radiation esophagitis(6-mV ray irradiated with a single large dose of 30Gy),synthesize different concentrations of nano-hydrotalcite combined ulcer fluid(1 times concentration,5 times concentration),and give Kangfuxin to the model rats respectively.solution,traditional Chinese medicine Kuiyangye,nano-hydrotalcite matrix,low-and high-concentration nano-Kuiyangye,and the body weight and food intake of rats were recorded and analyzed.After continuous intervention for 7 days,the samples were taken,and the esophagus tissue was fixed and sectioned for HE staining to evaluate the pathological damage of the esophagus tissue,and to screen the optimal dosage form for the treatment of radiation esophagitis in rats.2.2 Study on the mechanism of nano-Kuiyangye in the treatment of radiation esophagitis:The radiation esophagitis model rats were divided into normal group,model group,Kangfuxin liquid group,nano-matrix group,ulcer liquid group,and nano-ulcer liquid group.After continuous intervention for 7 days and 14 days,the samples were collected,and the arterial blood was centrifuged under anesthesia to obtain For serum,the esophageal tissues were taken for liquid nitrogen quick freezing and paraffin-fixed sectioning.To study the mechanism of nano-Kuiyangye in the treatment of radiation esophagitis through the three directions of inflammation,tissue repair and pain.①Nano-Kuiyangye inhibits inflammatory injury in rats with radiation esophagitis:Protein quantitative chip detects IFN-γ,TNF-α,IL-1β,MCP-1,IL-13,IL in rats with radiation esophagitis after 7 days of intervention with different drugs-10,IL-1α,IL-2,IL-4,IL-6 inflammatory factor expression differences,Western Blotting method was used to detect the local expression of inflammation-related pathway TLR4-MyD88-NF-κB in the esophagus tissue of rats with radiation esophagitis,ELISA method was used to verify the local expression of inflammatory factors IL-6,IL-10,TNF-α and IL-2 in esophageal tissue.To clarify the regulation mechanism of nano-Kuiyangye on various inflammatory factors and proteins in radiation esophagitis.②Nano-Kuiyangye promotes esophageal tissue repair in rats with radiation-induced esophagitis:slices were taken for 7 days and 14 days after continuous intervention for Masson staining to observe the expression of connective tissue fibers,and after Sirius red staining to observe the expression of collagen fibers with a polarized light microscope;Western Blotting method was used The expression levels of repair pathway-related proteins TGF-β1 p38MAPK,Smad4,FN,and EGF were detected;the expression levels of tissue repair factors α-SMA and HYP were detected by ELISA.To clarify the effect and regulation mechanism of nano-Kuiyangye on tissue repair of radiation-induced esophagitis.③Effect of nano-Kuiyangye on pain in rats with radiation esophagitis:ELISA method to detect pain factors prostaglandin-2(PGE-2),substance P(SP)and calcitonin gene-related peptide(CGRP)in serum of rats with radiation esophagitis To clarify the mechanism of nano-Kuiyangye inhibiting pain in rats with radiation esophagitis.2.3 Research on the mucosal adhesion and tissue drug absorption rate of nano-Kuiyangye:The model rats were randomly divided into ulcer liquid group and nano-ulcer liquid group,and were given daily intervention with ulcer liquid and nano-ulcer liquid.After 30min,60min,and 90min,the animals were killed and collected.A 3.0 cm esophagus was taken to obtain mucus on the surface of the esophagus and grinding liquid of esophageal tissue.Draw the "signal-concentration curve" of emodin,emodin methyl ether,chrysophanol,levoshikonin,and imperatorin standard products by high performance liquid chromatography-mass spectrometry(HPLC-MS),according to the signals of each effective substance in the sample Calculate the concentration according to the strength,and compare the remaining drug concentration on the surface of the esophageal mucosa and the drug concentration in the esophageal tissue of the nano-Kuiyangye and the Kuiyangye at different times after administration.Result:1 Clinical research:In terms of reducing the grading of inflammation and relieving pain in patients with radiation esophagitis,both the traditional Chinese medicine ulcer liquid and Kangfuxin liquid have good effects,and the traditional Chinese medicine ulcer liquid is non-inferior;the difference between the two groups is reflected in the nutritional status.Compared with before the intervention,it increased significantly,while the Kangfuxin liquid group decreased compared with before,suggesting that the food intake of patients after the intervention of traditional Chinese medicine ulcer liquid was higher than that of the Kangfuxin liquid intervention group,and the improvement of nutritional status was better than that of the Kangfuxin liquid group.2 Basic research2.1 Pharmacodynamic evaluation of nano-Kuiyangye in rats with radiation-induced esophagitis:Rats were irradiated with 30Gy to establish an animal model of radiation esophagitis.After the model was established,the weight of the rats was significantly reduced,and the food intake was significantly reduced.Esophageal tissue sections showed obvious esophageal tissue damage and inflammatory cell infiltration.After the intervention,the weight loss trend of the rats in each group did not change significantly,and there was no significant difference compared with the model group,indicating that the nano-Kuiyangye had no obvious effect on increasing the weight of the rats with radiation-induced esophagitis,which was consistent with the clinical observations.The food intake of rats with radiation esophagitis decreased significantly from the day of modeling compared with that before irradiation,and some rats stopped eating;while the total food intake of the high-concentration nano-Kuiyangye group was higher than that of the model group and other intervention groups within 7 days,indicating that the nano-ulcer Liquid tools can promote the recovery of eating in rats with radiation esophagitis,but only high-concentration nano-ulcer liquid has the effect.By comparing the pathological damage scores of the esophagus,the nano-hydrotalcite matrix group had no obvious pathological damage reduction compared with the model group,indicating that the simple nano-hydrotalcite matrix did not have a therapeutic effect;The pathological damage of the esophageal mucosa was significantly reduced,and the infiltration of inflammatory cells was also significantly reduced.Infiltration,the effect of reducing the damage of radiation esophagitis in rats is the best,and the effect is better than that of Kangfuxin liquid and traditional Chinese medicine ulcer liquid.2.2 Study on the mechanism of nano-Kuiyangye in treating radiation-induced esophagitis in rats:①Inhibition of inflammation:Through the chip detection of inflammatory factors in the esophageal tissue of irradiated esophageal rats,it can be seen that radiation exposure can cause changes in the expression of various inflammatory factors in esophageal tissue,which can make IL-2,IL-4,IL-13,MCP-1,IFN The expressions of-y and IL-1β increased,while the expression of IL-la and IL-10 decreased.nano-Kuiyangye has a regulatory effect on various inflammatory factors,can reduce the expression of pro-inflammatory factors IL-2,IL-6,TNF-α and increase the expression of anti-inflammatory factor IL-10;use Western Blotting to detect TLR4-MyD88-NF-κB inflammatory pathway protein It can be known that radiation exposure can increase the expression of inflammatory-related proteins TLR4,MyD88,and NF-κB in esophageal tissue;to suppress inflammation.The expression of inflammatory factors IL-2,IL-6;TNF-α,and IL-10 in esophageal tissue was verified by ELISA,and the change trend was consistent with the results of the inflammatory chip.It shows that the nano-Kuiyangye can regulate the levels of various inflammatory factors,and inhibit radiation esophagitis in rats through the TLR4-MyD88-NF-κB inflammatory pathway.②Promote tissue repair:After 7 days of continuous intervention,the repair effect of nano-Kuiyangye was not significant.The results of Masson staining and Sirius red staining indicated that the tissue structure of the esophagus in the model group was severely damaged after irradiation,and the connective tissue fibers and collagen in the submucosa were lost.Compared with the model group,there was no significant difference in the content of submucosal connective tissue fibers and collagen in the Kangfuxin liquid group,ulcer liquid group,and nano ulcer liquid group during 7 days of continuous intervention.The results of Western Blotting also confirmed this point.After radiation exposure,there was no significant difference in the expression of repair-related proteins TGF-β1,p38MAPK,Smad4,and EGF in esophageal tissue,while the expression of fibronectin FN,which represented the repair effect,decreased,indicating that the animal itself The ability to repair tissue damage is limited;after the intervention of nano-Kuiyangye,the expressions of TGF-β1,p38MAPK,and EGF increased compared with the model group,while the expressions of Smad4 and FN had no significant changes.It shows that the nano-Kuiyangye can increase the levels of various repair-promoting factors in the tissue repair process of radiation esophagitis,but the repair effect is not obvious after continuous intervention for 7 days.After 14 days of continuous intervention,the nano-ulcer liquid showed an obvious repairing effect.The results of Masson staining and Sirius red staining indicated that the tissue structure of the esophagus in the model group was severely damaged after irradiation,and the connective tissue fibers and collagen in the submucosa were lost.Compared with the model group,there was no significant difference in the fiber and collagen content of the submucosal connective tissue in the nano-matrix group;the fiber and collagen content in the Kangfuxin liquid group and the ulcer liquid group increased slightly,and the fiber and collagen content in the nano-ulcer liquid group increased significantly.The structure is close to normal esophageal tissue.The results of Western Blotting also verified this point.After the intervention of nano-Kuiyangye,the expression of FN increased significantly compared with the model group.The results of ELISA indicated that the change trends of α-SMA and HYP in rat esophagus tissue were consistent.Compared with the unirradiated normal group,the expressions of α-SMA and HYP in the model group were significantly decreased;after the intervention of nano-Kuiyangye,the expressions of α-SMA and HYP in the nano-Kuiyangye group were significantly higher than those in the model group;There was no significant difference compared with the model group.In conclusion,after 14 days of continuous intervention with nano-Kuiyangye,the loss of connective tissue fibers and collagen in esophageal tissue can be significantly restored under the microscope,and the expression of pro-repair factors TGF-β1,p38MAPK,and EGF can be increased,and the repair effect is obvious,and the expression of FN is significantly increased.The repairing effect of nano-ulcer liquid is better than Kangfuxin liquid and traditional Chinese medicine ulcer liquid,and the best time point to observe the repairing effect is 14 days.③Analgesia:Through ELISA detection on the serum of rats with radiation esophagitis,it can be known that radiation exposure can cause pain-related factors prostaglandin-2(PGE-2),substance P(SP),and calcitonin gene-related peptide in the serum of rats.The expression of(CGRP)increased,and the simple administration of nano-hydrotalcite matrix could not reduce the level of pain factors.nano-Kuiyangye could reduce the expression of three pain factors,and then play an analgesic effect.2.3 Study on the adhesion and absorption rate of nano-Kuiyangye in rats with radiation esophagitis The detection of five drug active ingredients in the esophageal mucus of rats with radiation-induced esophagitis showed that the adhesion time of the active ingredients in the nano-Kuiyangye to the esophagus surface was significantly longer than that of the traditional Chinese medicine Kuiyangye group,and the adhesion of the Kuiyangye was improved after nanometerization.At the time point of 90 minutes,the five active ingredients in the ulcer liquid intervention group were completely lost and the concentration could not be measured,while all the five active ingredients in the nano-ulcer liquid intervention group could be detected.Especially for the detection of emodin(Emodin),the concentration results indicated that the ulcer liquid group could not be detected at the three time points of 30min,60min,and 90min,and the drug residence time of the nano-ulcer liquid group was significantly longer than that of the ulcer liquid group;At the time point of component concentration,the concentrations of active components in the nano-Kuiyangye group were higher than those in the Kuiyangye group or the concentrations of the two were close.The detection of active ingredients in the esophageal tissue of rats with radiation esophagitis showed that after the intervention of nano-Kuiyangye,the concentration of active ingredients in the esophagus tissue was higher than that of the traditional Chinese medicine Kuiyangye group,and the absorption rate of active ingredients in the Kuiyangye was improved after nano-ulcerization.At each detection time point after administration,the concentration of active ingredients in the nano-Kuiyangye group in the esophagus tissue was higher than that of the Kuiyangye group or both concentrations were close.The concentration of emodin in the esophagus gradually decreased with time.Since 15 minutes,the emodin content in the esophagus of the nano ulcer liquid intervention group was higher than that of the traditional Chinese medicine ulcer oil group,and the concentration difference was the largest at 15 minutes;The gap was the largest,and the gaps at other time points were smaller,but from the overall trend,the concentration of active ingredients in the nano ulcer liquid group was still higher than that in the ulcer liquid group.Conclusion:1 Kuiyangye can effectively improve the symptoms of radiation esophagitis,reduce the grade of radiation esophagitis,relieve pain,improve the nutritional status of patients,and increase the level of prealbumin.2 Nano-Kuiyangye can effectively reduce the degree of inflammation in rats with radiation esophagitis,and promote the recovery of food intake in rats.3 In terms of molecular biological mechanism,the inhibition of inflammation by nano-Kuiyangye is related to the inhibition of the inflammatory response mediated by NF-κB inflammatory factors,and the inhibition of the increase of various pro-inflammatory factors such as IL-2,IL-6,and TNF-α.After 14 days of intervention with nano-Kuiyangye,the expressions of repair-related proteins such as TGF-β1 p38MAPK,FN,EGF,α-SMA,and HYP were significantly increased compared with those after 7 days of intervention,and the content of collagen and connective tissue fibers in the esophageal tissue structure was increased,thereby promoting the development of esophageal tissue.repair.The analgesic effect of nano-Kuiyangye may be related to the inhibition of PGE-2,SP,CGRP and other pain-related factors.4 In terms of the improvement of adhesion and absorption rate,the action time of the active ingredients in the nano-Kuiyangye adhered to the surface of the esophageal mucosa is prolonged,the adhesion of the nano-Kuiyangye is improved,and the absorption rate of the active ingredients in the ulcer fluid is improved after the nano-hydrotalcite is combined. |
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