| ObjectiveUlcerative Colitis (UC) is a kind of digestive tract disease characterized by chronic inflammation and ulcer formation in colon mucosa layer. UC is rated by the world health organization (WHO) as one of the modern diseases, which is difficult to be cured, with complex Etiology and many pathogcnesis links. Lung and bronchial lesions is one of parenteral manifestations in inflammatory bowel disease, with a high incidence, up to50%, according to literature reports. So far, there has been little research on the mechanism of lung injury in UC. UC is usually treated by amino salicylic acid treatment or cortex steroid, with insufficient effect. In this study, the UC animal model was induced by immune complex, TNBS and ethanol. Sodium houttuyfonate and diammonium glycynhizinate (DG) were used for animals, so as to explore apoptosis mechanism of lung injury in UC, and to reveal biological mechanism of lung injury in UC as well. Efficacy of sodium houttuyfonate and diammonium glycynhizinate was proved by their pathways, targets and the effects on apoptosis regulation,so as to provide an objective basis for the treatment of ulcerative colitis. And it may provide more reliable evidence for TCM treatment of UC and lung injury and further exploring theory of the lung and the large intestine being interioi-exteriorly related.Methods&ResultsI The characteristics of general status and morphological changes in rats with ulcerative colitis.Healthy male Wistar rats were induced by immune complex, TNBS and ethanol, divided into five groups:model group, western medicine group, sodium houttuyfonate group, diammonium glycynhizinate group, and sodium houttuyfonate plus diammonium glyeyrrhizinate group. Otherwise, there were30rats for normal group.0,2and4weeks after model establishment, general status and morphological changes were observed in rats. General living status of UC rats was worse than the normal rats, with significantly lower weight, breathing, shortness of breath and other symptoms of lung on week O. Pathology of lung tissue, such as lung interstitial inflammatory infiltrates, interstitial fibrosis, etc. was observed on all the time points. Lung injury might be caused by UC which is confirmed by animal models in this study. Therefore, lung injury is one of the parenteral performances of UC.2Apoptosis of inflammatory cell of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomerAccording to the principle of randomized, controlled experiment design, the rest of the model rats were randomly divided into five groups, namely model group, western medicine group, sodium houttuyfonate group, diammonium glycyrrhizinate group, and sodium houttuyfonate plus diammonium glycyrrhizinate group, after week0. Each treatment group was administrated by Chinese herb monomer or western medicine, while rats in normal group and model group were administrated by water as blank control. On the2nd and4th weekend, general condition of rats was observed. Otherwise, lung and colon tissue was taken for testing of indicators.2.1Apoptosis of lymphocytes of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomerFlow cytometry was used for dynamic testing apoptosis of lymphocytes of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomer, so as to discuss the relationship between abnormal lymphocytes apoptosis in peripheral blood and occurrence of UC, and to explore the mechanism of the TCM treatment of UC. Lymphocytes apoptosis rate in peripheral blood of rats with UC was significantly reduced. Combined the results of HE staining of colon tissue, it showed immune dysfunction of UC rats, structure damage of colon tissue, and infiltration of inflammatory cells. The results suggest that the lymphocyte apoptosis is involved in the pathogencsis of UC, and slow apoptosis of lymphocyte is closely related with occurrence of UC. Delay of lymphocytes apoptosis might be one of important pathological mechanisms, which may reflect immune function of UC rats.The Chinese and western medicine applied in the experiment may improve immune function of UC rats and alleviate symptoms by promoting apoptosis of lymphocyte. The improvement of lymphocyte apoptosis in peripheral blood rate by short-term application of diammonium glycyrrhizinate was better than that of sodium houttuyfonate, whose long-term effeet was better than short ones. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate for improving lymphocyte apoptosis was significantly better than single use of sodium houttuyfonate, which suggests superiority of treatment of UC from both lung and intestine.2.2Apoptosis of neutrophil of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomerFlow cytometry was used for dynamic testing apoptosis of neutrophil of peripheral blood in rats with ulcerative colitis and effect of Chinese herb monomer, so as to discuss the relationship between abnormal neutrophil apoptosis in peripheral blood and occurrence of UC, and to explore the mechanism of the TCM treatment of UC. Ncutrophils apoptosis was delayed in peripheral blood in model group, compared with normal one on the2nd week, while there was no difference in rats of neutrophils apoptosis between normal and model groups on the4th week, which suggest restoration of the delay. Considering general status of UC rats on the2nd week, such as mucinous or soft feces, ulcer and a large number of inflammatory cells infiltrated in colon tissue from images of HE staining, showed prolonged inflammation effect in blood or local by apoptosis inhibition of neutrophil, which suggest UC rats was in active period, with colon inflammation and ulcers. Otherwise, apoptosis of neutrophil was recovered on the4th week, while pathological damage of colon tissue was still existed, which showed UC rats was in remission on the4th week. It reveals that delayed apoptosis of neutrophil was one of important mechanisms of colon tissue injury in UC, and infiltration of neutrophil might be an important symbol of active UC. Changes in the rate of neutrophil apoptosis may provide certain basis for obsewation of UC and efficacy evaluation of drug. Otherwise, it may be a new ways for further study of neutrophil apoptosis and moderately control of apoptosis for clinical treatment of UC.In addition, neutrophil apoptosis was induced by use of sodium houttuyfonate and diammonium glycyrrhizinate, so UC rats got into remission in advance. Chinese herb monomer could reduce the inflammatory in a certain extent, so as to improve the mucosal injury, and to play a role in treatment of UC inflammation. Delayed apoptosis of lymphocyte and neutrophil was related with occurrence and development of ulcerative colitis. Chinese herb monomer may control apoptosis rats of lymphocyte and neutrophil in blood to achieve ease of UC.3Observation of apoptosis related protein, Caspase-3in UC rats and effect of Chinese herb monomer3.1Observation of apoptosis related protein, Caspase-3in colon tissue of UC rats and effect of Chinese herb monomerEnzyme linked immunoadsorption assay (ELISA) was used for dynamic observation of apoptosis related protein, Caspase-3in colon tissue of UC rats and effect of Chinese herb monomer, so as to confirm abnormal apoptosis in colon tissue, and to explore the effect of TCM treatment. Content of Caspase-3in colon tissue of UC rats was increased on the2nd and4th week, compared with normal rats, showing apoptosis acceleration of colon tissue existing in UC rats in a certain degree. Therefore, occurrence of UC may be related with excessive expression of Caspase-3in colon tissue of UC rats, which may induce apoptosis of colonic epithelial,and cause injury of colon tissue.In addition, SASP could improve the increased apoptosis of the colon tissue, but with insufficient long-term effect, while Caspase-3was decreased by Chinese herb in the colon. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate and separate use of diammonium glycyrrhizinale was superior to separate use of sodium houttuyfonate. 3.2Observation of apoptosis related protein, Caspase-3in lung tissue of UC rats and effect of Chinese herb monomerEnzyme linked immunoadsorption assay (ELISA) was used for dynamic observation of apoptosis related protein, Caspase3in lung tissue of UC rats and effect of Chinese herb monomer, so as to confirm abnormal apoptosis in lung tissue, and to explore the effect of TCM treatment. Content of Caspase-3in lung tissue of UC rats was increased, compared with normal rats on the2nd and4th week, showing apoptosis acceleration of lung tissue existing in UC rats in a certain degree. Therefore, occurrence of lung injury in UC may be related with excessive expression of Caspase-3in lung tissue of UC rats, which may induce apoptosis and cause injury of lung tissue.In addition, Chinese medicine was superior to SASP in improving the accelerated apoptosis in the lung. On the4th week Caspase-3of the lung was decreased significantly, with no evident changes on the2nd week by diammonium glycyrrhizinate. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate and separate use of sodium houttuyfonate could improve apoptosis of the lung in UC rats, with short and long-term effects. It suggests that the treatments represented by herbs could improve UC by decreasing Caspase-3level, and provide a new way to prevention and treatment of UC and its lung injury.4Observation of apoptosis related protein, Bcl-2/Bax in UC rats and effect of Chinese herb monomer4.1Observation of apoptosis related protein, Bcl-2/Bax in colon tissue of UC rats and effect of Chinese herb monomerWestern-bloting was used for dynamic observation of apoptosis related protein, Bcl-2/Bax in colon tissue of UC rats and effect of Chinese herb monomer. In normal group Bcl-2/Bax>1, cell apoptosis is relatively little. While in model group, Bax was continuously increased, and Bcl-2was decreased with Bcl-2/Bax<1, which suggested that cell apoptosis was in the ascendant. Therefore, apoptosis acceleration and abnormal level of Bcl-2/Bax in colon tissue may be one of the molecular mechanisms of colon injury in UCIn addition, Bcl-2was increased with decreased Bax and increased ratio of Bcl-2/Bax by both Chinese medicine and western medicine, which induced inhibition of apoptosis of colon tissue, so as to facilitate repair of the colon mucosa. Sodium houttuyfonate could increase the ratio of Bcl-2/Bax, and inhibit apoptosis of colon tissue, which was superior to SASP with long-term effect. Separate use of diammonium glycyrrhizinate and combined use of sodium houttuyfonate and diammonium glycyrrhizinate could increase the ratio of Bcl-2/Bax, so as to decrease the increased apoptosis of colon tissue. It suggest that Chinese medicine could improve apoptosis of colon tissue at both short and long term stages, which reveals characteristics and effectiveness of TCM treatment of UC from the lung and both from the lung and the intestine. 4.2Observation of apoptosis related protein, Bel-2/Bax in lung tissue of UC rats and effect of Chinese herb monomerWestern-bloting was used for dynamic observation of apoptosis related protein, Bcl-2/Bax in lung tissue of UC rats and effect of Chinese herb monomer. Bcl-2in lung tissue of UC rats was decreased on the2nd and4th week, while Bax was increased, with decreased Bcl-2/Bax, which suggested that increased apoptosis of lung tissue due to abnormal level of Bcl-2and Bax may be one of possible mechanisms of lung injury in UC. In addition, separate use of sodium houttuyfonate and diammonium glycyrrhizinate, and their combined use could increase Bcl-2and decrease Bax, with inhibited effect on apoptosis of lung tissue, which is better than SASP. Otherwise, combined use of sodium houttuyfonate and diammonium glycyrrhizinate was superior to their separate use. Lung disease was treated from the intestine and treated from both lung and intestine, which showed their special significances, providing theoretical basis for the theory of the lung and the large intestine being interior-exteriorly related.However, the pathogenesis of lung injury is very complicated. The lung and the large intestine being interior-exteriorly related is the result of a variety of substances and mechanisms. Therefore, study of Bcl-2/Bax for UC and lung injury is insufficient, which needs future comprehensive research.5Observation of apoptosis related protein, Fas/Fas-L in UC rats and effect of Chinese herb monomer5.1Observation of apoptosis related protein,Fas/Fas-L in colon tissue of UC rats and effect of Chinese herb monomerWestern-bloting was used for dynamic observation of apoptosis related protein, Fas/Fas-L in colon tissue of UC rats and effect of Chinese herb monomer. The content of Fas and Fas-L was increased in model group, compared with normal group, which suggest Fas/Fas-L may also participate in regulation of lung tissue apoptosis. Accelerated apoptosis of colon tissue may be related with abnormal increase of Fas and Fas-L.In addition, SASP, sodium houttuyfonate and diammonium glycyrrhizinate could decrease Fas or Fas-L in the colon tissue, while combined use of sodium houttuyfonate and diammonium glycyrrhizinate could both decrease Fas and Fas-L. It showed that both herbs and SASP could improve apoptosis of colon tissue, with each characteristic, while the effect of combined use of sodium houttuyfonate and diammonium glycyrrhizinate was better than that of SASP.The change of content of Fas and Fas-L and the intervention of drugs suggest that Fas and Fas-L may participate in pathological changes of colon tissue in UC rats, directly or indirectly. Otherwise, it showed pathways and targets by which herbs and therapies they represented may play a role.5.2Observation of apoptosis related protein, Fas/Fas-L in lung tissue of UC rats and effect of Chinese herb monomerWestern-bloting was used for dynamic observation of apoptosis related protein, Fas/Fas-L in lung tissue of UC rats and effect of Chinese herb monomer. The content of Fas and Fas-L was increased in lung tissue of UC rats, compared with normal group. Fas-L was decreased on the4th week in sodium houttuyfonate group and sodium houttuyfonate plus diammonium glycyrrhizinate group. There was no significant difference of Fas-L in lung tissue between western medicine group and model group, as well as diammonium glycyrrhizinate group. Tn addition, there was also no difference among other groups in Fas of the lung.6Observation of NF-κB in UC rats and effect of Chinese herb monomer6.1Observation of NF-κB in colon tissue of UC rats and effect of Chinese herb monomerEnzyme linked immunoadsorption assay (ELISA) was used for dynamic observation of NF-κB in colon tissue of UC rats and effect of Chinese herb monomer. The results showed that NF-κB, Bax, Fas, Fas-L and Caspase-3of colon were increased in UC rats on the2nd and4th week, with decreased Bcl-2and the ratio of Bcl-2and Bax. It suggested that NF-κB may regulat apoptosis of colon tissue on gene transcription levels through Bcl-2/Bax, and Fas/Fas-L system.On the2nd week, NF-κB of colon was decreased in sodium houttuyfonate plus diammonium glycyrrhizinate group, without improvement in other groups. On the4th week, NF-κB of colon was decreased in treatment groups except sodium houttuyfonate group. The results showed that both Chinese and western medicine could improve NF-κB of colon, while the effect of short-term use of SASP (sulfasalazine) was not obvious, and the short-term and long-term combined use of sodium houttuyfonate and diammonium glycyrrhizinate were both fairly good. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate was better than single use of which for intervention of NF-κB in colon tissue. It showed that treatment for both the lung and the large intestine was better than single one. In treatment of UC, compound of Chinese medicine was better than single application of Chinese herbs.6.2Observation of NF-κB in lung tissue of UC rats and effect of Chinese herb monomerEnzyme linked immunoadsorption assay (ELISA) was used for dynamic observation of NF-κB in lung tissue of UC rats and effect of Chinese herb monomer. The results showed that NF-κB, Bax and Caspase-3in lung tissue of UC rats was increased, while Bcl-2, the ratio of Bcl-2and Bax was decreased, compared with normal ones on the2nd week. It suggested that NF-κB may regulate apoptosis of lung tissue on gene transcription levels through Bcl-2/Bax system. No difference of NF-κB in lung tissue was shown between normal and model groups, while Fas, Fas-L and Caspase-3were still increased and the ratio of Bcl-2and Bax was decreased. It suggested that apoptosis of lung tissue still existed by recovery of lung injury.NF-κB in lung tissue was obviously decreased in sodium houttuyfonate group and sodium houttuyfonate plus diammonium glycyrrhizinate group on the2nd week, which suggested that single use of sodium houttuyfonate and combined use of sodium houttuyfonate and diammonium glycyrrhizinate was better than SASP. Otherwise, combined use of Chinese herbs was better than single use of diammonium glycyrrhizinate, which showed that treatment for both the lung and the large intestine was better than single one.ConclusionUC rats were used to evaluate effect of Chinese herb monomers, in order to explore apoptosis regulation mechanism of lung injury and biological mechanism of lung injury in UC. Otherwise, single and combined use of sodium houttuyfonate and diammonium glycyrrhizinate was evaluated for their effect, pathways and targets, which provide an objective basis for the treatment of ulcerative colitis. New ways and methods might be provided for clinical treatment of lung injury in patients. And it may provide more reliable evidence for TCM treatment of UC and lung injury and further exploring theory of the lung and the large intestine being interior-exteriorly related.1TNBS and ethanol with colonic mucosa tissue induced model rats were used in this experiment, which were relatively close to the clinical patients, with both systemic immune abnormalities and local inflammation pathological changes. The animal model is similar to pathological mechanism of UC in patients, with pathological mechanism of B cells and T cells induced immune response. Therefore, it is a suitable animal model for studying pathological mechanism and treatment effect of UC.2Delayed apoptosis of inflammatory cells may be related with UC, and may be important cause of continuous inflammation of ulcerative colitis.2.1Delayed lymphocytes apoptosis in peripheral blood was significant, which suggest that decreased rate of lymphocytes apoptosis may reflect immune function disorder in certain degree. Therefore, delayed lymphocytes apoptosis may be one of the important pathological mechanisms of UC. Thus, a new way to improve the immune function of patients with UC may be provided through intervention of lymphocyte apoptosis.2.2Apoptosis of neutrophil (PMN) in peripheral blood was delayed in UC rats at acute stages. Therefore, apoptosis of PMN was inhibited by prolonged existence in blood and inflamed lesions, which induced inflammatory injury of tissue, and may be one of the important mechanisms of injury of colon tissue in UC Its seepage may also be used as an important symbol of active UC.3Increased Bax, Fas, Fas-L, Caspase-3and NF-κB in colon tissue, with decreased Bcl-2, Bcl-2/Bax<1showed that accelerated apoptosis in the colon, which may be through apoptosis pathways, that is, NF-κB-Bcl-2/Bax and Fas/Fas-L systems Caspase-3, in order to preliminarily reveal possible apoptosis pathway in UC. Therefore, to increase Bcl-2. decrease Bax, Fas, Fas-L, Caspasc-3and NF-κB may inhibit accelerated apoptosis in colon tissue in certain degree, by interfering with the apoptosis pathways, so as to promote the repair of colonic mucosa and to provide evidence of molecular biology in treatment of UC.4In acute phase, Bax, Caspase-3and NF-κB were increased in lung tissue, while Bcl-2was decreased. In remission, NF-κB was improved, while other apoptosis related proteins were still abnormal, which suggested that lung injury was related with number of apoptosis regulatory proteins. Abnormal level of NF-κB, Bcl-2/Bax and Fas/Fas-L systems, and Caspase-3may induce apoptosis in lung injury, which may be one of possible mechanisms of lung injury in UC and lung disease due to the bowel. It may provide a new way to prevent and cure lung injury in UC.5The results showed thaf apoptosis in lung and colon was increased by possible pathways, that is, NF-κB-Bcl-2/Bax and Fas/Fas-L systems-Caspase-3. While apoptosis regulatory proteins may play different roles along with development of the disease. Theory of the lung and the large intestine being interior-exteriorly related was confirmed by UC animal model, in which tissue pathological injury of the lung and the colon with apoptosis acceleration of lung tissue existed. However, pathogenesis of UC and its lung injury is complicated, and relationship between the lung and the large intestine was correlated with a variety of substances. Thus, it needs future comprehensive research to reveal biological mechanisms of the lung and the large intestine being interior-exteriorly related, not only from the perspective of apoptosis mechanism.6Sodium houttuyfonate and diammonium glycyrrhizinate with their combined use, and SASP were overall evaluated through the effects on general condition of rats UC, pathology of the lung and the colon, peripheral inflammatory cells, apoptosis regulatory proteins, etc. with their own characteristics. Chinese medicine was generally superior to SASP in this study, which could improve apoptosis of the colon tissue mainly at acute stages, while Chinese medicine could both improve accelerated apoptosis of the colon and the lung tissue at acute and later stages. Combined use of sodium houttuyfonate and diammonium glycyrrhizinate was better than their separate use on improving accelerated apoptosis of the colon and the lung tissue, which reveals compound Chinese medicine is superior to separate use of one herb. In addition, sodium houttuyfonate could also improve apoptosis of the colon tissue in UC rats. Sodium houttuyfonate and diammonium glycyrrhizinate with their combined use, and SASP could improve apoptosis of the colon and the lung through one or more targets of the pathway, that is, NF-κB-Bcl-2/Bax、Fas/Fas-L-Caspase-3. Otherwise, a new way for prevention and cure of UC with its lung injury was provided through regulating and controlling target genes by Chinese medicine. Therefore, treatment from both the lung and the colon was superior to treatment from separate viscera, which showed that unique advantages of the theory of the lung and the large intestine being interior-exteriorly related. |
PDF Full Text Request