The Effects And Mechanisms Of Colla Corii Asini And Colla Corii Bovis On Mice With Chronic Obstructive Pulmonary Disease And Mice With Pulmonary Fibrosis

Background Chronic obstructive pulmonary disease(COPD)is a common respiratory system disease,which is characterized by persistent flow limitation.The COPD-related disability rate and mortality maintain high in the world.The COPD symptoms at acute exacerbation can be appropriately relieved after reasonable application of antibiotics and hormones;however,such measure can hardly prevent the COPD development progress.Pulmonary fibrosis is the progressive and irreversible lung function decline induced by a variety of reasons.Its morbidity shows an increasing trend in recent years,among which,the median survival time of idiopathic pulmonary fibrosis(IPF)is 2.9 years,with the 5-year survival of <50%.The pathogenesis of pulmonary fibrosis remains unclear at present,effective treatment is lacking,and its prognosis is poor.Currently,drugs such as glucocorticoids,immunosuppressors,immunomodulators and antioxidants have been frequently applied in clinic,but no remarkable clinical efficacy can be achieved.Moreover,they are associated with certain adverse reactions.Smoking is the major risk factor inducing COPD,which is also an important factor resulting in pulmonary fibrosis.Apart from respiratory system disease,smoking is also related to multiple diseases,such as cardiovascular and cerebrovascular diseases,as well as malignant tumors.According to epidemiological investigation results,the smoking rate shows a decreasing trend in recent years,but passive smoking remains extremely serious,and the yearly deaths of smoking-related diseases in China is growing.With the development of research,people gradually discover that immunity imbalance plays a key role in chronic airway inflammation,among which,the immune disorder of helper T cells(Th,CD4+)is regarded as one of the pathogenesis leading to multiple chronic respiratory system diseases,including COPD and asthma.Th cells have participated in multiple immune reactions of mucosal inflammation,which play a crucial role in the pathogenesis as a central link of immune regulation.Th cells can be classified into different subgroups based on the different functions of cytokines that they secrete,including Th1,Th2,Th17,Treg,Th9 and Th22 Of them,Th1 and Th2,as well as Th17 and Treg can inhibit each other during differentiation;therefore,their mutual balance is critical to maintain the immune homeostasis.Numerous studies have indicated that,adjusting the balance between Th1/Th2 and Th17/Treg subgroups can effectively control the genesis and development of chronic airway inflammation.Colla Corii Asini is a rare traditional Chinese medicine(TCM)in China,which has been used as medicine for over 2000 years since it is first recorded in the Holy Husbandman’s Classic on Roots and Herbs.Colla Corii Asini is originally made from cow hide,which has been changed to donkey hide due to historical reasons like war and has been used thereafter.cow hide gelatin is now named Colla Corii Bovis.Colla Corii Asini has extensive pharmacological functions,which is mainly used in clinic as a result of its blood replenishing effect.However,little is known about its Yin-nourishing and lung-moisturizing effect.It is recorded in ancients books that,Colla Corii Asini can treat chronic cough and consumptive lung disease(pulmonary fibrosis).In addition,it is used in modern clinic to treat chronic airway inflammatory diseases,such as pediatric cough,pulmonary tuberculosis,hemoptysis in bronchiectasia,pediatric asthma and chronic pharyngitis,and favorable therapeutic efficacy can be attained.Nevertheless,its mechanism of action in treating airway inflammation has not been illustrated yet.According to the Compendium of Materia Medica,Colla Corii Asini has superior effect on treating cough due to lung dryness than Colla Corii Bovis,but such theory has not been verified through experimental data yet.Therefore,intensively investigating the differences in the therapeutic effects and mechanisms of action between Colla Corii Asini and Colla Corii Bovis on treating chronic airway inflammation is of great significance to the development and quality control research on Colla Corii Asini as well as research on treating chronic respiratory disease using TCM.On the basis of the above background,this research first carried out a systemic review on the related records of the raw materials and functions of Colla Corii Asini and Colla Corii Bovis in ancient materia medica books.Afterwards,the airway inflammation mouse models were constructed using cigarette smoke exposure,so as to evaluate the therapeutic effects of Colla Corii Asini and Colla Corii Bovis on treating mouse airway inflammation.Subsequently,the pulmonary fibrosis mouse models were constructed based on literature review through cigarette smoke exposure combined with asbestos fiber inhalation,with an aim to investigate the suppression of Colla Corii Asini and Colla Corii Bovis on fibrosis.After determining the therapeutic effects of Colla Corii Asini,the helper T cell subgroup was used as the breakthrough point,to study whether Colla Corii Asini and Colla Corii Bovis could regulate the balance of Th1/Th2 and Th17/Treg,thus exploring the immune mechanism of Colla Corii Asini in treating airway inflammation and pulmonary fibrosis.Chapter 1 Herbal textual ResearchObjective Records regarding Colla Corii Asini and Colla Corii Bovis in ancient materia medica books were systemically reviewed,so as to study the correlation of pharmacological effects of Colla Corii Asini with raw material variation.Methods Ancient materia medica booklists mentioned in Chinese Medicine Philology were collected,and the representative literature records were screened after library and literature review.Meanwhile,literature at each time period was classified with distinct variation in Colla Corii Asini raw material as the time point,and the pharmacological effects of Colla Corii Asini were summarized and compared,so as to study the correlation of Colla Corii Asini raw material with its effects.Results There were generally 4 stages before the Colla Corii Asini raw material was changed from the original cow hide to the current donkey hide,and significant differences could be seen in the pharmacological effects of Colla Corii Asini at various stages.1.At the beginning of production to Tang Dynasty: Colla Corii Asini was made from cow hide,with the main effect of tonifying deficiency.2.Tang Dynasty: Colla Corii Asini could be made from both cow hide and donkey hide,and cow hide gelatin was mainly used for tonifying deficiency,while donkey hide gelatin was mainly used for dispeling wind.3.Ming Dynasty: Colla Corii Asini was mainly made from donkey hide,with the increased effect of tonifying Yin and clearing away the lung-heat.Meanwhile,the cow hide gelatin was also called Colla Corii Bovis,which was mainly used for traumatic injuries,carbuncle and pyogenic infections.4.Qing Dynasty: donkey hide was the unique raw material,while Colla Corii Asini made from cow hide was counterfeit goods.In addition,it was emphasized that the O well water was the essential condition to guarantee the quality of Colla Corii Asini,which was also the pharmacodynamic material basis for the cough-relieving and phlegm-eliminating effect of Colla Corii Asini.Conclusions Records of the effect on treating lung cough appear when the Colla Corii Asini raw material is officially changed to donkey hide.Moreover,more subsequent literature has pointed out and illustrated the functional differences between Colla Corii Asini and Colla Corii Bovis in treating airway inflammation based on the medicinal theory.These findings suggest that the effect of Colla Corii Asini on treating cough due to lung dryness may be related to its raw material.Chapter 2 Effects of Colla Corii Asini and Colla Corii Bovis on cigarette smoke induced airway inflammation in miceObjective To observe the therapeutic effects of Colla Corii Asini and Colla Corii Bovis on treating the cigarette smoke-induced airway inflammation mouse models.Methods 64 male C57BL/6 mice were randomly divided into control group,model group,Colla Corii Asini group and Colla Corii Bovis group.Airway inflammation mouse models were constructed using the cigarette smoke exposure method.In addition,the models were intervened with Colla Corii Asini and Colla Corii Bovis,and the model construction and drug intervention time was 16 weeks.The general status and weight changes were observed in mice,and the peripheral white blood cells were counted.8 mice were randomly selected from each group to determine the left bronchoalveolar lavage fluid(BALF)cell counts and cytokine contents.The upper lobe of right lung was fixed with 4% formaldehyde and prepared into HE staining pathological slices to observe the inflammation infiltration.The myeloperoxidase(MPO)activity in the tissue homogenate of middle and lower lobes of right lung was detected using ELISA kit.The levels of serum inflammatory media,including tumor necrosis factor-α(TNF-α),interleukin(IL)-8,IL-1β,IL-6 and IL-10,were determined using ELISA kit.Results The weight gain in model group was slow,which was markedly lower than that in normal group upon the end of modeling.In the meantime,both Colla Corii Asini and Colla Corii Bovis could effectively increase the mouse weight,and the weight gain velocity in Colla Corii Asini intervention group was faster than that in Colla Corii Bovis intervention group.However,difference in the mouse weight between administration group and control group was not statistically significant upon the completion of modeling.A large amount of inflammatory cell infiltration,pulmonary interstitial hyperemia edema and erythrocyte diapedesis could be observed in the lung HE stained sections in airway inflammation model mice,and the inflammation score was remarkably higher than that in control group.Compared with the model group,the airway inflammatory injury in Colla Corii Asini group was evidently improved,and the inflammation score was outstandingly reduced,while no obvious effects could be observed in Colla Corii Bovis group.The total white blood cell count in BALF of airway inflammation model mice was extremely markedly elevated,among which,the proportions of neutrophil and lymphocyte were increased,while that of macrophage was reduced.Colla Corii Asini could notably reduce the numbers and proportions of neutrophil and lymphocyte,while Colla Corii Bovis had lower capacity in recruiting neutrophil,resulting in indistinct reduction in the numbers of all cell types.The lung MPO activity in cigarette smoke-exposed mice was markedly elevated,while Colla Corii Asini intervention treatment could evidently reduce the MPO activity in mouse lung.In comparison,the lung MPO activity in Colla Corii Bovis group was also outstandingly lower than that in model group,but was still evidently higher than that in control group and Colla Corii Asini group.The protein expression levels of serum TNF-α,IL-1β,IL-8 and IL-6 in airway inflammation model mice were significant increased,but the IL-10 level was not markedly elevated.The serum IL-8,TNF-α and IL-1β levels in Colla Corii Asini group were markedly reduced,while the IL-10 level was significantly increased.In comparison,the IL-8 and TNF-α levels in Colla Corii Bovis group were evidently reduced,while IL-1β and IL-6 were not markedly changed,and IL-10 level was evidently elevatedbut lower than that in Colla Corii Asini group.Conclusions Colla Corii Asini has significant therapeutic effects on the airway inflammation in mice exposed to cigarette smoke,Colla Corii Bovis can effectively promote the weight gain in mice and alleviate part of the inflammatory media levels.But it can not markedly improve the inflammatory cell infiltration in lung,and has inferior overall effects to Colla Corii Asini.Chapter 3 Effects of Colla Corii Asini and Colla Corii Bovis on the helper T cell secreting pattern in cigarette smoke-exposed miceObjective To investigate the changes in the balance among lung and spleen helper T cell subgroups using cigarette smoke exposure Western blotting,as well as the intervention of Colla Corii Asini and Colla Corii Bovis on Th subgroup immune imbalance.Methods The residual mouse left lungs in each group after experiment in Chapter Two were taken and cut into pieces before collagenase digestion,followed by grinding into cell suspension.Subsequently,the mononuclear cell suspension was obtained through isolation with lymphocyte separating medium,and the changes in all Th cell subgroups in lung tissues were detected using flow cytometry after stimulation.The spleen was directly ground to prepare the cell suspension,and the changes in all Th cell subgroups in lung tissues were detected using flow cytometry after stimulation.The upper lobe of right lung was homogenized,and the expression of specific chemotactic transcription factors in all Th cell subgroups was detected using RT-PCR.The mouse BALF supernatant collected in Chapter Two was taken,and the expression of cytokines related to the differentiation of helper T cells was detected using ELISA kit.Results The proportions of CD3+ and CD8+ cells in lung of model group were markedly increased,and the proportions of Th1 and Th17 subgroups in helper T cells were elevated,while that of Treg subgroup was reduced.Colla Corii Asini could significantly suppress T cell activation,markedly lower the proportions of Th1 and Th17 subgroups while evidently elevate that of Treg subgroup.In contrast,Colla Corii Bovis group had lowered Th1 subgroup proportion,elevated proportions of Th2 and Th17 subgroups,and insignificantly changed Treg subgroup proportion.The m RNA transcription level of Th1 subgroup nuclear factor T-bet in model group was dramatically increased,that of Th2 subgroup nuclear factor GATA-3 was elevated,and the that of Th17 subgroup chemotactic transcription factor RORγt was markedly increased.In addition,the relative grey scale ratio of RORγt/Foxp3 m RNA transcription level was also remarkably raised.The transcription level of T-bet m RNA in lung tissue of Colla Corii Asini group was reduced,while those of GATA-3 m RNA and Foxp3 m RNA were dramatically elevated The transcription levels of GATA-3 m RNA,RORγt m RNA and Foxp3 m RNA in Colla Corii Bovis group were evidently increased.The IFN-γ and IL-17 A levels in BALF of model group were markedly increased,while the IL-5 and IL-10 levels were reduced.In comparison,the IFN-γ level was reduced in Colla Corii Asini group,while the IL-5 and IL-10 levels were increased,and IL-17 A level was lowered.The proportions of Th2 and Th17 subgroups were increased in spleen of model group,while that of Treg subgroup was markedly reduced.The Th1 subgroup proportion in Colla Corii Asini group was reduced,but the difference was not significant,while the Th2 and Th17 subgroups proportions were markedly reduced,while Treg subgroup proportion was dramatically increased.The proportions of Th2 and Th17 subgroups were not markedly changed in Colla Corii Bovis group,but the proportions of Th1 and Treg subgroups were enhanced.Conclusions Cigarette smoke-exposed mice are associated with the imbalance in the dominant differentiation of Th1 and Th17 subgroups.Colla Corii Asini can suppress the excessive protein expression of IL-6,IL-17 A and IL-23,as well as the m RNA expression of RORγt,whereas regulate the m RNA expression of IL-10 and Foxp3,thus regulating the differentiation balance of Th17/Treg subgroups.In addition,Colla Corii Asini can down-regulate the excessive protein expression of IL-12 and IFN-γ,as well as the m RNA expression of T-bet,thus suppressing the excessive differentiation of Th1 subgroup.In comparison,Colla Corii Bovis shows no obvious intervention on the immune imbalance in the dominant differentiation of Th1 and Th17 subgroups.As a result,it has inferior effects to Colla Corii Asini on suppressing inflammatory cell secretion and improving histopathological injury.Therefore,up-regulating the differentiation of helper T cells and the expression of IL-10 factor may be one of the mechanisms of action by which Colla Corii Asini treats the airway inflammation in cigarette smoke-induced mice.Chapter 4 Preventive effects of Colla Corii Asini and Colla Corii Bovis on pulmonary fibrosis in mice and the mechanisms of actionObjective To propose a novel method to construct the pulmonary fibrosis mouse model,and to evaluate the effectiveness of that method through lung pathological section observation and index determination.After successfully constructing the pulmonary fibrosis mouse model,the preventive effects of Colla Corii Asini and Colla Corii Bovis on the pathological changes such as fibrosis and airway remodeling were investigated.The lung helper T cell differentiation pattern in the model before and after drug intervention was analyzed,and the potential correlations of Colla Corii Asini and Colla Corii Bovis in intervening the helper T cell differentiation with their preventive effects on pulmonary fibrosis were also analyzed.Methods Model construction: 64 male C57BL/6 mice were randomly divided into control group,model group,Colla Corii Asini group and Colla Corii Bovis group.Cut tobacco from 5 cigarettes was mixed with 0.1g asbestos particles and placed into the passive smoking device for natural burnout,so that mice in model group and intervention groups were exposed in the environment full of the mixed smoke from cut tobacco and asbestos particles for once a day.2 sets were ignited continuously based on the above method each time,with the exposure time of about 30 min.Mice in Colla Corii Asini group and Colla Corii Bovis group were intervened with Colla Corii Asini and Colla Corii Bovis in the meantime of mixed smoke exposure,and the model construction and drug intervention time was 16 weeks.Model evaluation: 8 mice were randomly selected from each group upon the completion of modeling,and the left lungs were taken to prepare the HE stained pathological sections and Masson stained pathological sections.The pulmonary fibrosis degree in each group was rated according to the HE stained paraffin sections,and the Masson stained pathological section images were analyzed using IPP6.0 image processing software.Moreover,the collagen fiber area and smooth muscle thickness in each group were counted,and the bronchial smooth muscle index and collagen index were calculated.The middle and lower lobes of right lung were homogenized,and the protein contents of hydroxyproline,TGF-β1 and Smad3 in mouse lung tissues were detected using ELISA kit.The MMP-2 and MMP-9 m RNA expression in BALF of each group were detected using RT-PCR.The protein contents of IFN-γ,IL-4,IL-17 A and IL-10 in BALF of each group were determined using ELISA kit.The remaining mouse lung in each group was cut into pieces before collagenase digestion,followed by grinding into cell suspension.Subsequently,the mononuclear cell suspension was obtained through isolation with lymphocyte separating medium,and the changes in all Th cell subgroups in lung tissues were detected using flow cytometry after stimulation.Results HE stained pathological sections indicated type II alveolar epithelial cell hyperplasia in model group,along with increased ECM in lung tissue and alveolar septal thickening,which showed fibroid changes.In addition,difference in fibrosis score was statistically significant compared with the control group.Lung Masson stained pathological sections image analysis indicated that,collagen contents in bronchus and pulmonary mesenchyme in model group were markedly increased,and the difference was statistically significant compared with the normal control group.Meanwhile,the lung tissue hydroxyproline protein content determination results suggested markedly increased content of hydroxyproline in lung tissues,revealing the formation of pulmonary fibrosis pathological change.The above results indicated that,the pulmonary fibrosis mouse model could be successfully constructed after 16 weeks of exposure to smoke produced by the mixed ignition of cut tobacco of cigarette with asbestos particles.The collagen contents in the pulmonary mesenchyme were remarkably reduced in both Colla Corii Asini group and Colla Corii Bovis group,along with lower fibrosis score than that in the model group.In the meantime,the collagen fiber area,smooth muscle thickness,bronchial smooth muscle index and collagen index were reduced to some extent,among which,differences in collagen fiber area and smooth muscle thickness between Colla Corii Bovis group and model group were statistically significant At the same time,the above indexes indicated that,compared with Colla Corii Asini,Colla Corii Bovis could more remarkably prevent the formation of pulmonary fibrosisThe Th1 subgroup proportion in the lung of model group showed no marked change relative to the control group,but the proportion of Th2 subgroup was evidently elevated,and the Th1/Th2 balance had moved towards the Th2 polarity.Compared with the model group,Colla Corii Bovis intervention could up-regulate Th1 subgroup proportion in mouse lung,while remarkably down-regulate Th2 subgroup proportion.In comparison,the Th1 subgroup proportion in Colla Corii Asini group was elevated,whereas the Th2 subgroup proportion was reduced,but the differences were not significant.Compared with the control group,the lung Th17 subgroup proportion in model group was lower,the Treg subgroup proportion was notably higher,while the Th17/Treg ratio was dramatically lower than that in control group.After Colla Corii Asini intervention,the proportions of Th17 subgroup and Treg subgroup displayed no marked changes,while the Th17 subgroup proportion in Colla Corii Bovis group was significantly increased and the Treg subgroup proportion was evidently reduced.The expression level of IFN-γ in model group was markedly down-regulated,and the IFN-γ/IL-4 balance had moved towards IL-4.At the same time,the expression level of IL-17 A was reduced,while that of IL-10 was elevated.No significant changes were seen in IL-4 and IL-17 A in Colla Corii Asini group,the levels of IFN-γ and IL-10 were higher than those in the model group,but the differences were not significant.The IFN-γ level in Colla Corii Bovis group was outstandingly higher,the IL-17 A level was higher,and the IL-4 and IL-10 levels were markedly lower than those in model group.The MMP-9 transcription level in model group was remarkably down-regulated,while MMP-2 transcription level was elevated.Compared with the model group,the MMP-2 transcription level in Colla Corii Bovis group was down-regulated,and the MMP-9 transcription level was markedly up-regulated.In the meantime,the lung MMP-9 expression level in Colla Corii Asini group was up-regulated,but the up-regulation amplitude was smaller than that in Colla Corii Bovis group.Conclusions Smoke derived from the mixed ignition of cut tobacco from cigarette and asbestos particles can successfully induce the formation of pulmonary fibrosis in mice.The comparative results of pulmonary fibrosis score,collagen fiber area,bronchial smooth muscle thickness,bronchial smooth muscle index and hydroxyproline content in lung tissues suggest that,Colla Corii Asini displays superior effects to Colla Corii Bovis on inhibiting the formation of pulmonary fibrosis in mice.Further research indicates that,Th1/Th2 and Th17/Treg imbalance,as well as the abnormal expression of TGF-β1,MMP-2 and MMP-9 may participate in the genesis of pulmonary fibrosis.To be specific,Colla Corii Bovis can up-regulate Th1/Th17 subgroup differentiation and MMP-9 expression,restraint Treg subgroup differentiation,and down-regulate TGF-β1 and MMP-2 expression,thus effectively suppressing the genesis and development of pulmonary fibrosis.The Th subgroups secreting patterns in Colla Corii Asini group show no significant differences from those in model group,and differences in the expression of TGF-β1,MMP-2 and MMP-9 are not significant compared with the model group,thus,leading to inferior therapeutic effects to Colla Corii Bovis.However,the pulmonary fibrosis degree in Colla Corii Asini group is alleviated compared with that in model group,which may be related to the early reduction of inflammatory response.