| Objective:To deepen the understanding of TCM etiology and treatment theory of acute intestinal radiation injury by using local irradiation model,improve TCM compound prescription and evaluate its therapeutic effect on acute intestinal radiation injury,and explore its role and mechanism in promoting the repair of intestinal mechanical barrier and microbial barrier,so as to provide theoretical basis for improving the therapeutic effect of TCM on intestinal radiation injury.Method:1.Establishment of local irradiation intestinal mucosal injury model:120 male SD rats were randomly divided into 4 groups,12 in the control group,36 in each of the 10Gy,12Gy,and 14Gy groups.All rats except the control group were irradiated by a 60Co source（Institution of Radiation Medicine,Beijing,China）in the abdomen to establish an in vivo model of acute radiation enteritis.The irradiation range was from xiphoid process to pubic symphysis,and the rest parts were shielded by 5 cm thick lead plate.The doses of each irradiation group are 10,12,and 14 Gy respectively,and the dose rate is 102.5 c Gy/min.12 rats were randomly selected from each group to observe the survival;6 rats were randomly selected from each group on the4th,8th and 12th day after irradiation,blood was taken and sacrificed after anesthesia,and the organ damage was observed by naked eyes.After that,the small intestine was taken out,the serosal membrane,mucosa,and the sectioned surface were observed,and the histopathological changes of the small intestine were observed under a light microscope.The suitability of the modified model was evaluated according to the survival status of rats in each irradiation group2.Recognition of TCM etiology and treatment theory:Animals and groups are the same as the first part.The weight,food intake,defecation,activity,hair and death of rats in each group were observed.The esophagus,stomach,small intestine and large intestine were taken out to observe the serosa,mucous membrane and section.The histopathological changes of small intestine were observed under light microscope.According to the survival situation of rats in each irradiation group and the traditional Chinese medicine cold and heat,deficiency and excess,viscera,spleen and blood stasis,spleen and blood stasis of rats in each irradiation group In order to deepen the understanding of the etiology and treatment theory of acute intestinal radiation injury,and then adjust the prescription of traditional Chinese medicine.3.Evaluation of the therapeutic effect of Jiawei Liangxue Guyuan Decoction on acute radiation injury of intestinal mucosa:110 male SD rats were randomly divided into 6 groups:blank group（n=10）,model group（n=20）,glutamine group（n=20）and high-,medium-,and low-dose Chinese medicine groups（n=20）.1-7 days after modeling,the irradiation group and blank group were given normal saline 10 ml/kg,the glutamine group was given 0.3 g/100 g glutamine,the high,medium and low dose groups were given the same volume of Chinese medicine solution according to the dose of 18.8 g/kg·d,37.6 g/kg·d and 75.1 g/kg·d,once a day.Except for the blank group,5 surviving rats were selected from each group on the4th,8th and 12th day after irradiation.After pentobarbital sodium was injected intraperitoneally for anesthesia,the rats were killed by blood drawing.The changes of intestinal wall,lumen,mucosa and section characteristics were compared by observing the living and death conditions of food and water intake,activity,hair,bulbar conjunctiva,stool and body weight,The changes of intestinal radiation injury characteristics were observed under light microscope to evaluate the therapeutic effect of traditional Chinese medicine compound.4.Study on the effect and mechanism of Jiawei Liangxue Guyuan Decoction in promoting the repair of intestinal mucosal mechanical barrier and microbial barrier:using the above curative effect evaluation,the experimental specimens were made into intestinal pathological sections,and the destruction of intestinal mucosal tight junction was observed under electron microscope after staining,and the expression of NF-κB p65m RNA,TNF-αm RNA and IFN-γm RNA was detected by PCR in small intestinal mucosa;Western blotting was used to detect the expressions of MLCK,MLC,P-MLC,ZO-1,claudin-1 and occludin proteins in small intestinal mucosa.ELISA was used to detect the changes of s Ig A in small intestinal tissue and the levels of LPS and Dao in serum.The effects of the adjusted Chinese herbal formula on the repair of intestinal mucosal mechanical barrier and mlck-mlc pathway were evaluated.Another130 male SD rats were randomly divided into 7 groups,including 10 in blank group,20 in model group,20 in glutamine group,20 in Golden Bifid group and 20 in high,medium and low dose groups of traditional Chinese medicine.At 1-7 days after irradiation,the low,medium and high-dose Chinese medicine groups were given the corresponding dose of equal volume Chinese medicine decoction,the glutamine group was given 0.3g/100g glutamine,the Golden Bifid group was given 0.8g/kg/d Golden Bifid solution,the blank group and model group were given equal volume normal saline 10ml/kg,once a day.In addition to the blank group,5surviving rats were selected from each group on the 4th,8th and 12th day after irradiation.After pentobarbital sodium was injected intraperitoneally for anesthesia,blood was drawn and killed.The distal ileum 5cm away from the junction of jejunum and ileum was selected.After the intestinal wall was cut,80 mg of intestinal contents were collected with sterile pressure plate to detect intestinal flora,and serum was extracted to detect TNF-α,IL-6,IFN-γby ELISA.The changes of intestinal microflora and the correlation with inflammatory factors after radiation were analyzed,and the regulatory effects of Chinese herbal compound on microbial barrier and inflammatory factors were evaluated;The expression of TLR2,TLR4,TLR5 and TLR9 m RNA in small intestine was detected by PCR,and NF was detected by Western blot-κThe expressions of NFκB p65,My D88,COX2 and PGE2 were detected by Western blot.The expression of ZO-1,claudin-1 and occludin in small intestine was detected immunofluorescence method.Result:1.Evaluation of the model:the hyperactivity and polydipsia in each group were shorter than those in the previous model,and the degree of red eye and nose congestion was lighter;On the3rd to 5th day,rats in each group had watery stool,antifeedant and water repellent,perianal damp,dirty hair,which was consistent with the performance of the former model,and the decrease of body weight was less than that of the former model.Compared with the survival rate,all rats in 14Gy group died within 7 days after irradiation;The survival rate was 91.6%in10Gy group and 63.6%in 12Gy group.Macroscopic observation:edema,hematoma,mucosal necrosis and ulcer appeared in the intestinal mucosa of each group,which were consistent with those of the pre model,and intestinal flatulence and adhesion were more prominent.Microscopically:villus abscission and short alopecia appeared in the small intestine of each model group,crypt destruction was consistent with the former model,but the degree was reduced,but inflammatory cell infiltration was aggravated.2.Extended inspection of traditional Chinese medicine:in vivo observation,the irradiation group appeared irritability,excessive drinking,dry stool,red nose and conjunctival congestion1-2 days after irradiation.On the 3rd to 5th day,there were decreased intake and drinking water,refusal to eat,watery stool,bloody stool,abdominal distention,and decreased body weight;Anatomic observation showed multiple small bleeding foci,mucosal hematoma,necrosis,ulcer,intestinal adhesion,and a few intestinal perforation.Microscopically,intestinal villi were short,bald,atrophied,inflammatory cells infiltrated,crypts disappeared,and hemorrhage foci could be seen in lamina propria.On the 6th to 9th day,the hair color was dark and unsmooth,and the body weight and activity increased slightly;Anatomic observation showed that intestinal segment volvulus,adhesion,intestinal obstruction,accumulation of dark brown mucus in gastrointestinal cavity,ulcer and erosion still existed;Microscopically,the structure of intestinal mucosa recovered and inflammatory cell infiltration was less.From the 10th to the 12th day,the living observation showed that the rats’eating and drinking water returned to normal,but they were still curly and atrophied,with little movement,swollen abdomen,dry hair color,white claw nail,soft stool and increased body weight;Anatomic observation showed that intestinal adhesion,gas accumulation and obstruction decreased,and intestinal tissue was transparent and easy to break;Microscopically,the mucosal structure was restored,but there was thrombosis in the small vessels of lamina propria.The above characteristics have the conditions to use the eight principles syndrome differentiation,viscera syndrome differentiation,Qi and blood syndrome differentiation and Wei Qi Ying blood syndrome differentiation analysis methods.3.Evaluation of Jiawei Liangxue Guyuan Decoction in the treatment of acute radiation injury of intestinal mucosa:typical manifestations of radiation injury of intestinal mucosa appeared 3-5 days after irradiation,such as watery stool,severe bloody stool,and significantly reduced body weight.The diarrhea of each treatment group was relieved,with the glutamine group being the most obvious,followed by the high and medium dose Chinese medicine group,and the low dose group was ineffective.The two-week survival rate of the model group was about 45%,and all the treatment groups were improved to a certain extent,especially in the high and medium dose groups of traditional Chinese medicine,which increased to 75%,followed by glutamine group and low dose group of traditional Chinese medicine,with the survival rate of about 60%.The body weight of rats in each group decreased significantly after irradiation（P<0.01）.On the 4th day after irradiation,the body weight of rats in the high and medium dose groups was significantly higher than that in the model group（P<0.01,P<0.05）.There was no significant difference between the glutamine group and the model group.On the8th and 12th day,there was no significant difference between the treatment groups and the model group.Organ anatomy observation:on the 4th day after irradiation,obvious edema,submucosal hematoma,mucosal necrosis and ulcer appeared in the model group,which were alleviated in the high and medium dose groups,followed by glutamine group and low dose group.Light microscope observation:after irradiation,the villi of intestinal mucosa epithelium of rats in each group were shortened,abscission and necrosis in varying degrees,especially in the model group.The structure of high and medium dose groups was relatively complete.4.Study on the effect and mechanism of Jiawei Liangxue Guyuan Decoction in promoting the repair of intestinal mucosal mechanical barrier and microbial barrier:the electron microscope observation of the experimental specimens showed that the distribution and density of intestinal epithelial microvilli in the blank group were more uniform,arranged in order,and the structure of tight junctions between cells was very complete and tight,while the microvilli on the surface of cells in the model group were reduced,with different lengths,Uneven distribution and scattered arrangement;The results showed that the tight junction structure was incomplete,partially broken,loose,desmosomes density decreased and gap junction widened significantly.The high-dose group and glutamine group had the best effect on the restoration of villus morphology and tight junction,followed by the medium dose group and the low-dose group.On the 4th day after irradiation,s Ig A expression level in small intestine of all irradiation groups was significantly lower than that in blank group,especially in model group（P<0.01）.s Ig A expression level in high-dose group was significantly higher than that in model group（P<0.01）,followed by glutamine group（P<0.05）;On the 8th day after radiation,the middle dose group was higher than the model group（P<0.05）,but there was no significant difference between the glutamine group and the low dose group and the model group;On the 12th day after radiation,only the high dose group was higher than the model group（P<0.05）.The expression of LPS in serum:on the 4th day after irradiation,LPS in each irradiation group increased significantly（P<0.01）.Compared with the model group,the expression of LPS in high-dose group decreased most significantly（P<0.01）,followed by medium and low-dose groups and glutamine group（P<0.05）;On the 8th day after radiation,the high dose group was significantly lower than the model group（P<0.01）,followed by glutamine group（P<0.05）;On the 12th day after radiation,there was no significant difference between the treatment groups and the model group.The level of Dao in serum was significantly increased on the 4th day after radiation,especially in the model group（P<0.01）.The level of Dao in the medium and high dose groups of traditional Chinese medicine was lower than that in the model group（P<0.05）.There was no significant difference between the low dose group of traditional Chinese medicine and the glutamine group and the model group;On the 12th day after radiation,there was no significant difference between the treatment groups and the model group.On the 4th day after irradiation,compared with the blank group,the NF-κB p65,TNF-αand IFN-γm RNA expression in the model group was significantly increased（P<0.01）;compared with the model group,the NF-κB p65,TNF-αand IFN-γm RNA expression in the high,medium and low dose groups of traditional Chinese medicine and glutamine group were significantly down regulated（P<0.05 or P<0.01）.Compared with the blank group,the protein expressions of MLCK,MLC and P-MLC in the model group were significantly increased,and the protein expressions of ZO-1,occludin and claudin-1 were significantly decreased（P<0.01）;compared with the model group,the protein levels of MLCK,MLC and P-MLC in the high-dose group and glutamine group were significantly decreased（P<0.01）,and the protein levels of ZO-1,claudin-1 and occludin were significantly increased（P<0.05 or P<0.01）.The richness of out flora was evaluated by shapom index.The richness of blank group was the highest.The difference between model group and blank group was the most significant on the 4th day after radiation（P<0.01）.PCo A analysis showed that the difference of bacterial community structure between model group and blank group was the largest on the 4th day after radiation,and the difference of bacterial community structure between model group and blank group gradually narrowed with time.At the class level,the relative abundances of bacilli（3.1%）and Bacteroidetes（15.7%）decreased significantly on the 4th day after radiation,while the relative abundances of gamma Proteobacteria（38.2%）and clostridia（34.8%）increased slightly.On the 8th and 12th day after radiation,the relative abundances of gamma Proteobacteria decreased gradually,while the abundances of bacilli,clostridia and bacteroidia increased,and the proportion of bacteria approached normal;at the genus level,the proportion of Lactobacillus（0.8%）and muribaculate（10%）in the model group decreased significantly on the 4th day after irradiation.The dominant bacteria were Escherichia Shigella（36.8%）,blautia（7.3%）,flavonifractor（4.2%）and others（10.9%）.On the 8th day after irradiation,the dominant bacteria were Escherichia Shigella（20.3%）and Enterococcus（21.6%）,Lactobacillus（18.3%）and Lactobacillus（10.9%）On day 12 after irradiation,the proportion of Lactobacillus（17.6%）and muribacullace（13.7%）decreased slightly,and the proportion of Escherichia Shigella decreased to 15.6%.On the 4th day after irradiation,the intestinal microflora of all treatment groups recovered to varying degrees,especially glutamine and Chinese medicine medium dose group had the best inhibitory effect on Escherichia Shigella（P<0.01）;On the 8th day after irradiation,Golden Bifid group had the best inhibitory effect on Escherichia Shigella（P<0.01）,followed by high and low dose groups of traditional Chinese medicine（P<0.05）;12 days after irradiation,the inhibition of Escherichia Shigella was the best in Golden Bifid and high and low dose groups of traditional Chinese medicine,followed by medium dose group of traditional Chinese medicine（P<0.05）,and there was no significant difference in other groups.After irradiation,pro-inflammatory factor TNF-α,IL-6 and IFN-γincreased in varying degrees（P<0.05）,and gradually decreased with the extension of irradiation time,but they were still higher than that in the blank group;IL-10 decreased significantly after irradiation（P<0.01）,and increased gradually with the extension of irradiation time,but it was still lower than the normal level.On the 4th day after irradiation,the levels of TNF-α,IL-6 and IFN-γin glutamine group and Chinese medicine groups were significantly lower than those in control group The expression level of IL-10 was lower than that of model group（P<0.05）.The expression level of IL-10 was also increased in glutamine group（P<0.05）.There is a significant correlation between the intestinal flora and inflammatory factors.The abundance of probiotic flora represented by Lactobacillus is negatively correlated with the expression of pro-inflammatory factors TNF-α,IL-6 and IFN-γ（P<0.05）.It is positively correlated with the expression of anti-inflammatory factor IL-10（P<0.05）,and harmful bacteria represented by Escherichia-Shigella are positively correlated with the expression of pro-inflammatory factors TNF-α,IL-6 and IFN-γ（P<0.01）,negatively correlated with the expression of IL-10,an anti-inflammatory factor（P<0.01）.Compared with the blank group,TLR2,TLR4,TLR5 and TLR9 m RNA expression levels were significantly increased in the model group（P<0.01）;compared with the model group,the glutamine group,the Jinshuangqi group and high,medium and low doses of traditional Chinese medicine group were significantly down-regulated（P<0.01）.Compared with the blank group,NF-κB p65,My D88,COX2 and PGE2 protein expression levels were significantly increased in the model group（P<0.01）;compared with the model group,the high-dose traditional Chinese medicine group and the Golden Bifid group significantly decreased（P<0.05or P<0.01）;My D88 and COX2 protein expression in the high-dose group of traditional Chinese medicine decreased（P<0.01）.Conclusion:1.Evaluation of local irradiation model:60Co-γThe male SD rat model of acute radiation-induced intestinal injury prepared by 12gy single abdominal irradiation has the characteristics of prolonged survival,typical pathological changes,deficiency and excess of cold and heat,viscera Qi and blood syndrome differentiation system,which is more suitable for the basic theoretical research and prevention research of TCM etiology and treatment.2.Further understanding of the theory of etiology and syndrome differentiation and treatment:through repeated use of extended inspection and a variety of dialectical analysis methods for dynamic observation and analysis,it is considered that the etiology of acute intestinal radiation injury is still heat toxin.In the early stage,it is"heat toxin is abundant in the interior,and Qi and blood are burnt",and in the middle stage,it is"heat burns blood,Spleen Yang is not vibrating,and dampness toxin is mixed";According to this,the new strategies of prevention,treatment and rehabilitation were put forward,which included"clearing heat and cooling blood,supporting yang and spleen to remove dampness,tonifying qi and Yang,cooling blood to consolidate Yin,removing dampness and detoxification,nourishing yin and Yang,strengthening spleen to remove dampness,removing blood stasis and dredging collaterals to promote rehabilitation",On the basis of the original prescription,the modified Liangxue Guyuan Decoction strengthened the function of"greatly tonifying vital energy,benefiting qi and absorbing blood,strengthening spleen and stopping diarrhea".3.Evaluation of the curative effect of the adjusted traditional Chinese medicine in the treatment of acute radiation injury of intestinal mucosa:Jiawei Liangxue Guyuan decoction can still effectively reduce the radiation injury of intestinal mucosa,improve the quality of life of rats after radiation,reduce diarrhea,and improve the short-term survival rate,which has the basis of expanding and deepening research to clarify its mechanism of action.4.The effect and mechanism of the adjusted traditional Chinese medicine prescription in promoting the repair of intestinal mucosal mechanical barrier and microbial barrier:（1）Jiawei Liangxue Guyuan decoction can protect intestinal mucosal mechanical barrier by promoting the recovery of intestinal mucosal epithelial tight junction and microvilli after radiation injury,reducing intestinal mucosal permeability and promoting s Ig A secretion,and its mechanism is related to the inhibition of MLCK-MLC pathway.（2）Jiawei Liangxue Guyuan decoction can promote the recovery of intestinal flora richness and diversity after acute intestinal mucosal radiation injury.It has obvious inhibitory effect on Shigella and other harmful bacteria,thus plays a protective role on microbial barrier,and can balance inflammatory mediators and reduce pro-inflammatory factor TNF-α,IL-6 and IFN-γ.The specific mechanism may be related to the inhibition of TLRs pathway. |
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