We first analyzed the performance of the rhesus monkeys after PQ poisoning. Rhesus monkeys behaved normally in the initial stage, but within 12–30 h, they exhibited one or more of the following clinical symptoms: lethargy, decreased activity, vomiting, abdominal distension, and drooling. Rhesus monkeys treated with 25 mg/kg and 40 mg/kg PQ survived until the end of follow-up, while rhesus monkeys treated with 80 mg/kg PQ died after 1 week (Supplementary Fig. 1).

The concentration of PQ in rhesus monkey blood was the highest at 12 h. At that point, the PQ concentration in the blood of those treated with 25 mg/kg was 170.83 ng/mL (Fig. 1A). The PQ concentration in the blood of those treated with 40 mg/kg was 330.33 ng/mL. The PQ concentration in the blood of those treated with 80 mg/kg was 966.75 ng/mL at 12 h and 114.56 ng/mL at 36 h (Fig. 1A, Supplementary Fig. 2A). Over time, the concentration of PQ in the blood decreased until it was undetectable.

Blood results in a rhesus monkey model with pulmonary fibrosis induced by oral PQ at 25, 40, or 80 mg/kg. B Changes in arterial oxygen saturation (SaO2) during modeling. C Changes in serum potassium concentration. D Counts of white blood cells, neutrophils, and lymphocytes in blood. The B–D legend is the same as A. PQ paraquat. Data are presented as the mean ± SEM (error bars). n = 3 per group. Between-group comparison at 25 mg/kg vs. 40 mg/kg, *p < 0.05. Between-group comparison at 25 mg/kg vs. 80 mg/kg, #p < 0.05

The blood pressure of the rhesus monkeys did not change significantly. The arterial oxygen saturation (SaO2) of the rhesus monkeys treated with 25 or 40 mg/kg PQ decreased briefly at 60 h. However, the SaO2 of the rhesus monkeys treated with 80 mg/kg decreased at 12 h (Fig. 1B, Supplementary Fig. 2B). Hypokalemia is a negative prognostic marker in paraquat poisoning [23]. Therefore, we analyzed the serum potassium concentration. There was a slight fluctuation in serum potassium concentration in rhesus monkeys treated with 25 or 40 mg/kg PQ. The serum potassium of rhesus monkeys treated with 80 mg/kg PQ increased to 5.3 mmol/L at 1 week (Fig. 1C, Supplementary Fig. 2C). The proportion of white blood cells and neutrophils in their blood increased in the early stage of PQ poisoning and returned to normal 1 week later (Fig. 1D, Supplementary Fig. 2D).

Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) in the blood of rhesus monkeys treated with 25 or 40 mg/kg PQ increased from 12 to 60 h and then gradually decreased. In rhesus monkeys treated with 80 mg/kg PQ, AST increased significantly at 12 to 48 h, but ALT did not change significantly (Fig. 2A, Supplementary Fig. 3A). Markers of renal function, such as creatinine (CREA), uric acid (UA), and urea, of rhesus monkeys treated with 40 mg/kg PQ was higher than normal in the first 4 weeks. Rhesus monkeys treated with 25 or 80 mg/kg PQ showed only small fluctuations in renal function (Fig. 2B, Supplementary Fig. 3B). Creatine kinase (CK), hydroxybutyrate dehydrogenase (HBDH) and lactate dehydrogenase (LDH) are relevant markers for cardiac injury, hepatic or muscle injury, and hemolysis. These markers were significantly increased in the early stage and essentially returned to normal 2 weeks later (Fig. 2C, Supplementary Fig. 3C).

The results of liver function, kidney function, and cardiac enzyme profile in the blood of three rhesus monkeys. A Changes in ALT and AST. B The contents of CREA, UA, and UREA in renal function in serum with the passage of time. C Change trend of related indices of cardiac enzyme profile. The B–D legend is the same as in A. AST aspartate aminotransferase, ALT alanine aminotransferase, CREA creatinine, UA uric acid, CK creatine kinase, HBDH hydroxybutyrate dehydrogenase, LDH lactate dehydrogenase. Data are presented as the mean ± SEM (error bars). n = 3 per group. Between-group comparison at 25 mg/kg vs. 40 mg/kg, *p < 0.05. Between-group comparison at 25 mg/kg vs. 80 mg/kg, #p < 0.05

The CT images of rhesus monkeys induced with 25 mg/kg PQ were evaluated as follows. At 12 h, pneumomediastinum appeared in the lungs, with exudative changes in both lungs. At 60 h, the right lung was atelectatic, and there was a small amount of exudate in the middle lobe. After 1 week, the lung markings were disorganized, with left subpleural changes and thickening of the interlobar fissures (Fig. 3A). At 4 weeks, ground-glass lesions appeared in the lungs. Diffuse ground-glass lesions were more obvious at 12 weeks. The CT results revealed that pulmonary fibrosis may have developed in rhesus monkeys after 4 weeks (Fig. 3B, Supplementary Table 1).

Computed tomography (CT) imaging results of rhesus monkeys. A The results of rhesus monkeys during the early stages of PQ induction. B In rhesus monkeys treated with 25 mg/kg PQ, ground-glass lesions after 4 weeks of follow-up. C CT of rhesus monkeys treated with 40 mg/kg PQ during weeks 2–8. Arrows are annotated as follows: in the 25 mg/kg group, exudative changes in both lungs at 12 h; exudation in the upper left lung at 36 h; right atelectasis at 60 h; right lung shrinkage and poor light transmittance at 2 weeks; ground-glass lesions at 12 weeks. In the 40 mg/kg group, pneumothorax ①, mediastinal emphysema ② occurred at 12 h; pneumothorax ①, mediastinal emphysema ②, lung apex consolidation exudation ③ at 60 h; pneumothorax occurred at 4 weeks; atelectasis occurred at 5 weeks

Lung CT images of rhesus monkeys treated with 40 mg/kg PQ showed obvious mediastinal emphysema at 12 h. At 60 h, there was pneumothorax and hemorrhage in the right lung. The apex of the upper lobe of the lung showed consolidation exudation, compressed atelectasis, pleural effusion, and mediastinal emphysema aggravation. Mediastinal emphysema disappeared at 1 week (Fig. 3A). At 4 weeks, pneumothorax and mediastinal emphysema appeared again in the lungs, and there was a small bilateral subpleural effusion. Atelectasis was also observed at 5 weeks, and there was no pulmonary fibrosis detected by CT until 8 weeks (Fig. 3C, Supplementary Table 1). Rhesus monkeys treated with 40 mg/kg PQ showed a more severe progression compared to the clinical pulmonary fibrosis course, but no obvious fibrosis was seen by CT until 8 weeks. In addition, CT images of rhesus monkeys treated with 80 mg/kg PQ also did not show pulmonary fibrosis.

We further observed the pathological changes in the lung during the induction of pulmonary fibrosis by histopathology of lung biopsy samples. Lung biopsy tissue samples of rhesus monkeys treated with 25 mg/kg PQ showed extensive cellular thickening of the interalveolar septa, inflammatory cell infiltration, and alveolar structural disorder or destruction in the early stages. The results of Masson's trichrome staining and Sirius red staining also indicated that PQ may have induced excessive collagen deposition in lung tissue (Fig. 4, Supplementary Fig. 4, Supplementary Table 2). After 1 week, it still displayed inflammatory cell infiltration, increased interstitial cells with a fibroblastic appearance, and fibrogenesis. After 4 weeks, lung tissue exhibited fibrosis and obvious inflammatory infiltration (Fig. 5, Supplementary Fig. 4). We used α-SMA as an immunohistochemical marker of alpha-smooth muscle actin, a feature of myofibroblasts. The results showed significant collagen deposition at 12 weeks (Fig. 5). The α-SMA staining was consistent with the results of Masson staining (Fig. 5). The expression of α-SMA was high at 4–12 weeks in the rhesus monkeys treated with 25 mg/kg. Even at 12 weeks, pulmonary fibrosis was still present, which was consistent with the CT results.

Lung histopathological results of three PQ-poisoned rhesus monkeys. A The results of HE, Masson and Sirius red staining in lung tissue. B Pathological results of rhesus monkeys poisoned with 25 mg/kg PQ for 12 weeks. C The results of rhesus monkeys poisoned with 40 mg/kg PQ for 8 weeks. D and E was the semi-quantitative statistical result of collagen on Masson staining and Sirius red staining, respectively. Scale bars = 50 μm

The IHC results of CD68, F4/80, MAC 387, and α-SMA in lung tissue. Between-group comparison at 25 mg/kg vs. 40 mg/kg, *p < 0.05

The lungs of the rhesus monkeys treated with 40 mg/kg PQ showed diffuse hemorrhage at an early stage. Collagen deposition appeared at all time points after 1 week (Fig. 4, Supplementary Table 2). From 1 to 2 weeks, inflammatory cells infiltrated and exudated obviously in the alveolar cavity (Fig. 5). Masson and Sirius red staining showed collagen deposition up to 8 weeks. The α-SMA staining results were consistent with the Masson and Sirius red staining results.

After 1 week, rhesus monkeys treated with 80 mg/kg PQ died of poisoning. Lung histopathology showed mild inflammation and no obvious collagen deposition. However, alveolar edema and collapse were observed. Hemorrhagic foci were observed in the lung tissue. High doses of oral PQ also caused intestinal mucosal cell shedding. The liver and kidneys were also damaged (Fig. 6).

Autopsy lung pathology results of 80 mg/kg PQ-poisoned rhesus monkeys at 1 week (A) and HE staining of other organs (B). Scale bars = 200 μm

We found that oral administration of 25 mg/kg PQ in rhesus monkeys was more consistent with the development of pulmonary fibrosis. To understand the early changes in the lung at this dose, we replicated PQ poisoning in rhesus monkeys with an oral dose of 25 mg/kg. Then, BALF and lung tissues from transbronchial lung biopsies were collected. The concentration of PQ in BALF was 46.7 ng/mL (Fig. 7A). In the BALF, white blood cells increased at 12 h and gradually returned to normal (Fig. 7B). The biochemical indices of the rhesus monkeys treated with 25 mg/kg peaked at 36 h and then returned to normal (Fig. 7C). Lung tissue sections obtained by transbronchial lung biopsy displayed extensive cellular thickening of the interalveolar septa, interstitial edema, and inflammatory cell infiltration. The results demonstrated that the alveolar structure was damaged, with extensive collagen deposition at 36 h and 60 h (Fig. 7D).

BALF results and transbronchial lung biopsy histopathology of rhesus monkeys treated orally with 25 mg/kg PQ. A PQ concentration in BALF. B Classification of white blood cells, neutrophils, and lymphocytes in BALF. C Changes in liver function, kidney function, and myocardial enzyme profile in BALF. D Pathology results of transbronchial lung biopsy. BALF bronchoalveolar lavage fluid, PQ paraquat. Scale bars = 50 μm. n = 3 per group