Objective

2

To explore the possible mechanism of

Yiqi Yangyin Huoxue Tongluo Formula

（益气养阴活血通络方， YYHTF） in treating idiopathic pulmonary fibrosis （IPF） based on CD40-CD40L system.

Methods

2

Totally， 96 Wistar rats were randomly divided into blank group， model group， pirfenidone group， and YYHTF group， with 24 rats in each group. IPF rat model was established by intratracheal injection of bleomycin hydrochloride in all groups except for the blank group. On the next day after modeling， the pirfenidone group was given pirfenidone capsules by sequential gavage； the dosage was 62.5 mg/kg each day on the 1st to 7th day， while 125 mg/kg on the 8th to 14th day， and 187.5 mg/kg on the 15th to 28th day. The YYHTF group was given YYHTF granules at the dosage of 18 g/kg by gavage. The blank group and model group were given 10 ml/kg of normal saline by gavage. All interventions were given once a day for 28 days. On the 7th， 14th， and 28th day since the next day after modelling （8 rats were measured at each timepoint of each group）， hematoxylin eosin （HE） staining was used to observe the pathological changes of lung tissue； the contents of hydroxyproline （HYP） in lung tissue of rats in each group were detected by alkaline hydrolysis， and the expression levels of plasminogen activator inhibitor （PAI）， coagulation factor Ⅸ （FⅨ）， coagulation factor Ⅶ （FⅦ） and platelet factor 4 （PF-4） in serum were detected by enzyme-linked immunosorbent assay； the expression levels of CD41 and CD62P in platelets were detected by flow cytometry， and the protein levels of CD40 and CD40L in lung tissue of rats were detected by western blotting.

Results

2

HE staining showed normal in the lung tissue of the rats in the blank group. In the model group， pathological results showed that the alveolar septum were widened， and the inflammatory cells in the lumen were gathered with obvious edema in the pulmonary interstitium on the 7th day； collagen fibers hyperplasia was seen in the pulmonary interstitium on the 14th day； the alveoli was gradually atrophied， and a large number of collagen fibers were deposited on the 28th day. There were increased contents of HYP in lung tissue， as well as the expression levels of CD41 and CD62P in platelets， the serum PAI， FⅨ， FⅦ and PF-4 expression levels， and protein levels of CD40， CD40L in lung tissue in the model group than in the blank group measured on the same timepoint on the 7th， 14th and 28th days （

P

＜0.01）. Compared to those measured at the same timepoint in the model group， the infiltration of inflammatory cells in the pirfenidone group and YYHTF group was significantly improved and the fibrosis was significantly reduced according to the pathological morphology； the contents of HYP in lung tissue， the expression levels of CD41 in platelets， expression levels of serum PAI， FIX， FⅦ and PF-4， and protein levels of CD40 and CD40L in lung tissue of rats in two medication groups significantly decreased to varying degrees on the 7th， 14th and 28th days （

P

＜0.05 or

P

＜0.01）. Compared to those in the pirfenidone group， the contents of HYP in lung tissue， and the serum expression levels of serum PAI， FⅨ， FⅦ and PF-4 in the YYHTF group decreased on the 7th， 14th and 28th days， while the expression levels of CD41 and CD62P in platelet decreased on the 7th and 28th days， and the expression level of CD40 in lung tissue decreased on the 28th day （

P

＜0.05 or

P

＜0.01）.

Conclusion

2

The mechanism of YYHTF in the treatment of IPF may be related to the inhibition of platelet activation and CD40-CD40L system.