| Backgrounds&PurposePulmonary fibrosis is a chronic,progressive,and fatal lung disease.Alveolar inflammation,persistent damage and excessive repair of the alveoli are caused by a variety of reasons and lead to the deposition of interstitial fibers in the lung,resulting in localized scar tissue.This process is irreversible and progresses rapidly.It can be caused by dust,drugs and so on.There are still some patients with idiopathic pulmonary fibrosis but the pathogenesis is not clear.The treatment of pulmonary fibrosis is limited,and there is no radical cure except pulmonary transplantation.Median survival from diagnosis to death is only 3-5 years.Pirfenidone is an orphan drug only marked the indications of drugs on pulmonary fibrosis in the instructions.But its clinical use is limited because there are many side effects in the digestive system and the nervous and mental system.There is a lack of drugs for pulmonary fibrosis,on the other hand,of the drug targets have not been found,therefore,that better curative effect,pulmonary fibrosis therapeutics more safety and possible drug targets has been one of the direction of pharmaceutical research personnel.The main clinical manifestations of pulmonary fibrosis appeared cough expectoration and later progressive dyspnea.It is characterized by chronic,recurrent attacks.The main clinical manifestations of pulmonary fibrosis appeared cough expectoration and later progressive dyspnea.It is characterized by chronic,recurrent attacks.TCM classified it into "pulmonary flaccidity" and"pulmonary arthralgia".Through the long-term research,the main pathogenesis of pulmonary fibrosis is phlegm,blood heat and blood stasis,blocking the lung collaterals,causing the lung to be destroyed,and the Qi motor block.In clinic,the treatment of clearing away heat and toxic material,promoting blood circulation and removing blood stasis has certain preventive and therapeutic effect on pulmonary fibrosis.Polygonum cuspidatum has a long history of medicinal use and it is recorded in the traditional Chinese Medicine Classics such as "Theory of drug properties","Supplement to the Herbal" about pharmaceutical application.Polygonum cuspidatum is the dry root and rhizome of Polygonum cuspidatum Sieb.et Zucc.It tastes bitter and has a slight cold and belongs to liver,gallbladder and lung meridian.It tastes bitter and has a slight cold and belongs to liver,gallbladder and lung meridian.It has the efficacy of remove dampness through diuresis,clearing away heat and toxic material,dispersing blood stasis and relieving pain,relieving cough and eliminating phlegm.Modern studies have shown that Polygonum cuspidatum has an anti-lung fibrosis effect.Polydatin is one of the main components of Polygonum cuspidatum in anti-pulmonary fibrosis.In order to further clarify the effect of polygonin in pulmonary fibrosis and to explore its possible mechanism,find potential drug targets,the experimental mouse model of bleomycin induced pulmonary fibrosis,observe the treatment effect of polydatin from the overall level of animal,observe the lung tissue transforming growth factor(TGF-β)of the intervention effect so as to provide more evidence for the application of polydatin in the treatment of pulmonary fibrosis.Method:1.Replication of Bleomycin Induced Pulmonary Fibrosis In a Mouse ModelTo compare the advantages and disadvantages of the two models of pulmonary fibrosis by intratracheal instillation and intraperitoneal injection of bleomycin(BLM),and determine the best mode of preparation.Healthy male C57BL/6 mice,aged 7 weeks and conventional feeded for 7 days,were randomly divided into 11 groups,5 in each group.Intratracheal administration of 6 groups were 3 days group,7 days group,14 days group,21 days group,28 days group and blank control group.Administrating the animals of bleomycin saline solution by intratracheal instillation,dose equivalent to bleomycin 2 mg/kg by weight.Stipulate the starting date of the experiment is the 0th day.After administration,on the third day,the seventh day,the fourteenth day,the twenty-first day,the twenty-eighth day sacrificed the animal after get their eyeball blood,collecting serum and bronchoalveolar lavage fluid,lung tissue.The blank group was treated the same volume as normal saline.Intraperitoneal injection of 5 groups,7 days group,14 days group,21 days group,28 days group and blank control group.Stipulate the experiment began in the th day.Administrating them with 0.1 mg/mL intraperitoneal injection of bleomycin saline solution,dose equivalent to bleomycin 1.5 mg/kg by weight every other day,lasted two weeks,totaled 8 times,and the 7 day group was injected 4 times.The animals were sacrificed on the seventh,fourteenth,twenty-first and twenty-eighth days respectively.The animals were sacrificed after collecting serum,bronchoalveolar lavage fluid and all lung tissues.The blank group and the control group were injected with equal volume normal saline.The best molding condition was determined by pathological characteristics of lung tissue.2.The effect of polydatin on lung fibrosisBased on the previous experimental results,the model of pulmonary fibrosis was reproduced by intratracheal instillation of bleomycin saline.Healthy male C57BL/6 mice,7-8 weeks old,adaptive feeding for 7 days,were randomly divided into 5 groups,normal control group,model group,positive drug pirfenidone group,polydatin prevention group,polydatin treatment group,5 rats in each group.Normal control group received intratracheal instillation of sterilized normal saline,while other groups received 2 mg/kg of body weight by intratracheal instillation of sterile saline solution of bleomycin.Positive drug control group,polydatin prevention group were administrated by gavage(171mg/kg weight)the second day after modeling to the 28th day.Polydatin treatment group started from the seventh day after model(171 mg/kg weight)while the control group and model group mice were gavaged from the second day after operation with the same volume of pure water to the 28th day.The protective effect of polydatin on pulmonary fibrosis was evaluated according to the pathological changes of Masson staining;Hydroxyproline content indicates the change of collagen in pulmonary fibrosis;Inflammatory cell count in bronchoalveolar lavage fluid predicts changes in inflammatory response during pulmonary fibrosis;The content of TGF-β 1/2/3 in lung tissue of mice was detected by mouse TGF-β protein chip.Result:1.Masson staining showed that 7 days after model establishment bleomycin intratracheal instillation group inflammatory cells increased,sporadic collagen fibers increased;a small area of collagen fiber deposition appeared in the 14th day.There were many plaques of collagen fiber deposition in the 21th day,and the area was larger;The degree of fibrosis in the 28th day is similar to the 21th day and slightly increased;Obvious progressive process of pulmonary fibrosis can be seen from the 7th day to the 28th day.After intraperitoneal injection the mice were contracted with pulmonary fibrosis.Pathological findings showed that on the 7th day,inflammatory cells such as macrophages,lymphocytes and neutrophils were distributed;the interstitial lung of the 14th day showed signs of increased collagen;on the twenty-first day,a small quantity of collagen fibers appeared in the lung stroma;the 28th day were similar with the 21th day,but there were no plaques of collagen fiber deposition.2.Compared with the lung tissue in the blank control group,the lung tissue structure of the model control group was seriously damaged.The pathological manifestations of severe pulmonary fibrosis were presented.The lung fibrosis of polydatin prevention group was obviously reduced,presented as sporadic small area of collagen fiber deposition.Compared with the prevention group,the improvement of lung fibrosis in the polydatin treatment group was weaker than that in the prevention group,and the area of fibrosis was larger than that of the prevention group,Compared with the model control group,it has an improving effect.The positive control drug pirfenidone reduced the degree of pulmonary fibrosis,and the therapeutic effect of polydatin treatment group had no significant difference.The results of hydroxyproline content and bronchoalveolar lavage fluid count showed that the result was not correlated with the degree of pulmonary fibrosis(P>0.05).The results of lung tissue TGF beta protein microarray showed TGF-βincreased during early phase and fell back at later phase in lung tissue after bleomycin administrated.Conclusion:The results showed that intratracheal instillation of bleomycin could induce obvious pulmonary fibrosis,which was similar to the clinical characteristics of pulmonary fibrosis.Polydatin can significantly reduce the degree of lung fibrosis in mice,and has potential therapeutic effect on pulmonary fibrosis. |
PDF Full Text Request