Objective:

2

To investigate the common mechanisms among collagen-induced arthritis (CIA)

bleomycin (BLM)-induced pulmonary fibrosis

and CIA+BLM to evaluate the therapeutic effect of triptolide (TP) on CIA+BLM.

Methods:

2

Thirty-six male Sprague-Dawley rats were randomly assigned to 6 groups according to a random number table (n=6 per group): normal control (NC)

CIA

BLM

combined CIA+BLM model

TP low-dose (TP-L

0.0931 mg/kg)

and TP high-dose (TP-H

0.1862 mg/kg) groups. The CIA model was induced by intradermal injection at the base of the tail with emulsion of bovine type Ⅱ collagen and incomplete Freund's adjuvant (1:1)

with 200 μL administered on day 0 and a booster of 100 μL on day 7. Pulmonary fibrosis was induced via a single intratracheal injection of BLM (5 mg/kg). The CIA+BLM model combined both protocols

and TP was administered orally from day 14 to 35. After successful modeling

arthritis scores were recorded every 3 days

and pulmonary function was assessed once at the end of the treatment period. Lung tissues were collected for histological analysis (hematoxylin eosin and Masson staining)

immunohistochemistry

measurement of hydroxyproline (HYP) content

and calculation of lung coefficient. In addition

HE staining was performed on the ankle joint. Total RNA was extracted from lung tissues for transcriptomic analysis. Differentially expressed genes (DEGs) were compared with those from the RA-associated interstitial lung diseases patient dataset GSE199152 to identify overlapping genes

which were then used to construct a protein-protein interaction network. Hub genes were identified using multiple topological algorithms.

Results:

2

The successfully established CIA+BLM rat model exhibited significantly increased arthritis scores and severe pulmonary fibrosis (

P

<

0.01). By intersecting the DEGs obtained from transcriptomic analysis of lung tissues in CIA

BLM

and CIA+BLM rats with DEGs from rheumatoid arthritis-interstitial lung disease patients (GSE199152 dataset)

50 upregulated and 44 downregulated genes were identified. Through integrated PPI network analysis using multiple topological algorithms

IGF1 was identified as a central hub gene. TP intervention significantly improved pulmonary function by increasing peak inspiratory flow (

P

<

0.01)

and reduced lung index and HYP content (

P

<

0.01). Histopathological analysis showed that TP alleviated alveola

r collapse

interstitial thickening

and collagen deposition in the lung tissues (

P

<

0.01). Moreover

TP treatment reduced the expression of collagen type Ⅰ and α-SMA and increased E-cadherin levels (

P

<

0.01). TP also significantly reduced arthritis scores and ameliorated synovial inflammation (

P

<

0.05). Both transcriptomic and immunohistochemical analyses confirmed that IGF1 expression was elevated in the CIA+BLM group and downregulated following TP treatment (

P

<

0.05).

Conclusion:

2

TP exerts protective effects in the CIA+BLM model by alleviating arthritis and pulmonary fibrosis through the inhibition of IGF1-mediated EMT.