| Part Ⅰ To evaluate the state of Qi deficiency and blood stasis in experimental IPF rats and reveal the intervention characteristics of Danggui Buxue TangObjective: To evaluate the characteristics of traditional Chinese medicine(TCM)syndromes in idiopathic pulmonary fibrosis(IPF)rats and observe the intervention characteristics of invigorating Qi and activating blood medicine.Methods: 60 male specific pathogen free(SPF)level SD rats(weighted180-200g)were randomly divided into sham group,model group,prednison group and Danggui Buxue Tang group.A rat model of pulmonary fibrosis was induced by intratracheal instillation of bleomycin(5 mg/kg).The second day after surgery,the rats in the sham and the model group were given deionized water(1 m L/100 g)and the rats in the Danggui Buxue Tang group were given sedimentary solution of granules(8.1 g/100 g/d),while the rats in the prednison group were given sedimentary solution of prednison(5 mg/100 g/d).All the rats were orally administered once per day for 14 consecutive days.The general biological characterization of rats and pathology morphology change of lung tissue,percent survival of the rats were observed.Changes in body weight and food intake were monitored.Weight-bearing swimming time and characteristics of hemorheology were determined.An evaluation questionnaire of syndrome score was conducted to assess the Qi deficiency and blood stasis syndrome of rats.Contrast-enhanced laser speckle imaging of the dorsal auricular vein in rats were observed,and the quantities of microcirculatory blood perfusion units(MBPU)were calculated.Results:1.Observation of the general state of rats in each groupThe rats in the sham group recovered to the preoperative state in 4 days after the operation,with active behaviors and sensitive reactions,white hair,smooth breathing,and smooth claw armor,nasal tip and tail tip.The rats in the model group were more prone to lying and idling,their hair was withered and yellow,and it was shriveled and easy to fall off.They were short of breath and had asthma,and their nose,mouth and claw armor were darker and darker.Although the conditions of the rats in the Danggui Buxue Tang group and prednison group were still dumbness,the eye movement was relatively flexible,and the reaction was slightly delayed,but the movement was still more sensitive,and the nose,lips and claw armor were slightly dark purple.Although the hair was yellow,but still shiny,and asthma can be heard occasionally.2.Observation of pathological morphology of lung tissue in ratsRats in the sham group had complete lung tissue structure,alveolar cavity and bronchial cavity were clean,with little sky-blue collagen deposition and inflammatory cells.In the model group,the lung tissue structure was destroyed,alveoli was collapsed and fused,and alveolar wall was thickened.A large amount of sky-blue collagen and fibroblasts were deposited in the pulmonary interstitium,and obvious inflammatory cell infiltration was observed in the alveolar cavity and pulmonary interstitium.The alveolar structure of the treated group was relatively complete.Inflammatory cell infiltration,collagen and fibroblast deposition were reduced,and alveolar wall was thinner than that of the model group.3.Comparison of body weight,food intake,survival rate,weight-bearing swimming time,MBPU,scores of Qi deficiency and blood stasis syndrome and changes of hemorheologyCompared with the sham group,the rats in the model group had a substantially lower body weight,food intake,percent survival,weight-bearing swimming time,and MBPU(P < 0.05 or P < 0.01),while viscosity of low,medium and high incision and the syndrome score of Qi deficiency and blood stasis were up-regulated(P<0.01).Compared with the model group,the body weight,food intake,percent survival,weight-bearing swimming time,and MBPU of rats in the two treated group were increased significantly(P < 0.01).Additionally,and the viscosity of low,medium and high incision in the whole blood and the score of Qi deficiency and blood stasis syndrome were decreased significantly(P < 0.05 or P < 0.01).Compared with the prednison group,the rats in the Danggui Buxue Tang group experienced significant increases in weight-loaded swimming time and MBPU(P < 0.05 or P < 0.01),as well as the whole blood viscosity of low,medium and high incision and the score of Qi deficiency and blood stasis syndrome(P < 0.05 or P < 0.01).Summary:1.Intratracheal infusion of bleomycin can induce an IPF rat model;2.Qi deficiency and blood stasis is an important TCM syndrome characteristic of IPF rats;3.Danggui Buxue Tang can significantly improve the state of Qi deficiency and blood stasis in rats,suggesting that invigorating Qi and activating blood is an important treatment option for IPF;4.Danggui Buxue Tang is better than prednison in improving the overall condition of the body.Part Ⅱ: Study on the formation of material basis of Qi deficiency and blood stasis in experimental IPF rats and explore the intervention mechanism of Danggui Buxue TangObjective: To observe the effect of Danggui Buxue Tang on inflammatory factors in lung tissue of IPF rats,and reveal the possible mechanism of intervention of tonifying Qi and activating blood circulation on IPF,thus investigating the possible material basis of Qi deficiency and blood stasis in IPF rats.Methods: Animals,modeling and drug administration methods were the same as those in part I.Lung function changes were analysed.Pulmonary index and cell count in bronchoalveolar lavage fluid were determined.Semi-quantitative analysis of hematoxylin eosin(HE)staining and Masson staining were conducted.Colorimetric method,radioimmunity method and alkaline solution were applied to detect the myeloperoxidase(MPO)activity,hyaluronan(HA)and hydroxyproline(HYP)content in lung tissue,respectively.Immunohistochemical staining,real-time fluorescent quantitative PCR and enzyme-linked immunosorbent assay were used to detect the expression of interleukin 1β(IL-1β),IL-6 and tumor necrosis factor α(TNF-α)in lung tissue.Western blotting was experienced in the detection of CollagenⅠ,Collagen Ⅲ,α-smooth muscle actin(α-SMA)protein expression in lung tissue.Results:1.Comparison of pulmonary function of rats in each groupCompared with the sham group,elastance,tissue elastance,newtonian airway resistance and total respiratory resistance of the model group were significantly increased,while the compliance and tissue damping were decreased considerably(P < 0.01).Compared with the model group,the elastance,tissue elastance,newtonian airway resistance and total respiratory resistance of the rats in the two treated group were all decreased significantly,while the compliance and tissue damping increased significantly(P < 0.01).2.Comaprison of inflammatory damage in lung of rats in each groupCompared with the sham group,the Szapiel score,cell classification count of bronchoalveolar lavage fluid,MPO activity and expression of IL-1β,IL-6 and TNF-α were significantly increased(P < 0.05 or P < 0.01).Compared with the model group,the Szapiel score,cell classification count of bronchoalveolar lavage fluid,MPO activity and expression of IL-1β,IL-6 and TNF-α of the treated group significantly decreased(P < 0.05 or P < 0.01).3.Comaprison of the signs of fibrosis in lung of rats in each groupCompared with the sham group,the lung index,Ashcroft score,collagen volume fraction,HA and HYP contents,Collagen Ⅰ,Collagen Ⅲ,α-SMA levels were significantly increased(P < 0.05 or P < 0.01).In the two treatment group rats,the lung index,Ashcroft score,collagen volume fraction,HA and HYP contents,Collagen Ⅰ,Collagen Ⅲ,α-SMA levels were significantly decreased(P < 0.05 or P < 0.01).Summary:1.Immune inflammatory response is an important pathological link of IPF.Excessive activations of inflammatory cells and release of inflammatory factors are important mechanisms leading to collagen deposition and extracellular matrix aggregation,which contributed to pulmonary dysfunction;2.Danggui Buxue Tang can reduce collagen deposition and extracellular matrix aggregation by inhibiting the activation of inflammatory cells and the release of inflammatory factors,thus improving lung function in IPF rats;3.Inflammatory cytokines(IL-1β,IL-6 and of TNF-α)might be the important substance basis for the state of Qi deficiency and blood stasis in IPF rats,as well as the biological targets for the role of Danggui Buxue Tang in invigorating Qi and activating blood circulation.Part Ⅲ Danggui Buxue Tang inhibits TLR4/NLRP3 signaling pathway to reduce lung inflammation and injury in IPF ratsObjective: To reveal the mechanism of Danggui Buxue Tang in inhibiting inflammatory reaction and reducing signs of fibrosis,and to explain the molecular biological mechanism of Danggui Buxue Tang in promoting Qi and activating blood circulation,to clarify the signal regulation mechanism of Qi deficiency and blood stasis in IPF rats.Methods: Animals,modeling and drug administration methods were the same as those in part I.Expression of Toll like receptor 4(TLR4),myeloid differentiation factor88(My D88),nuclear factor kappa-B(NF-κB),Nucleotide-binding oligomerization domain-like-receptor family pyrin domain3(NLRP3),apoptosis-associated speck-like protein containing CARD(ASC),cysteinyl aspartate specific proteinase 1(Caspase-1)were detected by real-time quantitative PCR and Western blotting.Results: TLR4,My D88,NF-κB,NLRP3,ASC,Caspase-1 protein and gene expression were significantly increased in the model group compared with the sham group(P < 0.01).Compared with the model group,TLR4,My D88,NF-κB,NLRP3,ASC,Caspase-1 protein and gene expression levels in the treatment group were significantly decreased(P < 0.05 or P < 0.01).Summary: Danggui Buxue Tang inhibited lung inflammation and fibrosis in IPF rats by inhibiting TLR4/NLRP3 signaling pathway,which may be an important target for Danggui Buxue Tang to promote Qi and blood circulation.TLR4/NLRP3 signaling pathway may be an important regulating mechanism of the state of Qi deficiency and blood stasis in IPF.Conclusion:1.Qi deficiency and blood stasis is an important symptom of IPF,and invigorating Qi and activating blood circulation is an important treatment for this disease,which has advantages in improving the overall state of the body;2.Inflammatory factors may be an important substance basis for the appearance of Qi deficiency and blood stasis in IPF.Invigorating Qi and activating blood circulation method can alleviate degrees of fibrosis by inhibiting inflammatory responses;3.Invigorating Qi and activating blood circulation method can protect the lung tissue of IPF by inhibiting the activation of TLR4/NLRP3 signaling pathway.Qi deficiency and blood stasis of IPF may be regulated by TLR4/NLRP3 signaling pathway. |
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