Protective Effects Of Pterostilbene On Paraquat-induced Pulmonary Fibrosis And The Underlying Mechanisms

Background Administration of paraquat (PQ) can induce lung toxicity and the process of PQ lung toxicity can be clearly divided into two stages:acute lung injury and pulmonary fibrosis. The toxic effects of PQ are due to its ability to induce oxidative stress, inflammatory and fibrotic reactions. Redox cycling and subsequent generation of reactive oxygen species (ROS) is the main mechanism of PQ toxicity.Sirtl is a crucial cellular survival protein. ROS can inhibit Sirtl activity by evoking oxidative modifications on its cysteine residues. Sirt1can stimulate the expression of antioxidants, which have the effect of anti-oxidative stress.Pterostilbene (PTS), found in grapes, blueberries and rosewood, has many pharmacological activities including analgesia, anti-aging, anti-cancer, anti-inflammatory, antioxidant and anti-proliferative functions.The present study has two parts, the first part aims to research whether Sirtl is involved in acute lung injury and pulmonary fibrosis induced by PQ; according to the results of the first part, the second part aims to research whether pterostilbene can regulate the expression of Sirtl protein and inhibit the generation of ROS to protect lung from injury induced by PQ.Method In the first part:acute lung injury mouse model was established by a bolus injection of PQ (30mg/kg) intraperitonealy. Mice were divided into2groups:control and PQ. Eight hours after PQ injection, total cell numbers, total protein concentration, and the level of TNF-α and IL-6in bronchial alveolar lavage fluid (BALF) were determined. The histopathological change was detected by HE staining. The level of ROS was detected by using dihydroethidium (DHE) staining in lung tissues. Cellular apoptosis was assessed by TUNEL assay and Caspase-3activity in lung tissues. Protein expression of Sirtl was detected by Western Blot in lung tissues. Pulmonary fibrosis mice were established by a bolus injection of paraquat (20mg/kg) intraperitonealy. Mice were divided into2groups:control and paraquat. Twenty-one days after injection, testing indexes and methods were the same as acute lung injury model. Moreover, the collagen deposition was measured by Masson assay.In the second part:mice were divided into6groups:control, PQ, vehicle, PQ (20mg/kg) plus PTS (20mg/kg), PQ (20mg/kg) plus PTS (40mg/kg), and PTS (40mg/kg). After a single dose of PQ (20mg/kg, intraperitonealy), PTS was chronically administrated to mice for21days. Testing indexes and methods were the same as pulmonary fibrosis model.Results The results of the first part:1. Compared with normal control group, the lung tissue of acute lung injury mice showed alveolar septal thickness, collapse of alveoli, hemorrhage, and extensive inflammatory cell infiltration in the alveolar space and septum; the lung tissue of pulmonary fibrosis model mice showed destruction of alveolar structure, as evidenced by the thickness of alveolar septa, loss of alveolar spaces and infiltration of inflammatory cells. Masson’s trichrome staining showed collagen deposition.2. Compared with normal control group, total cells and total protein in BALF of mice in the PQ group was significantly increased. 3. Compared with normal control group, cell apoptosis ratio and Caspase-3activity in lung tissues was significantly increased in the PQ group.4. Compared with normal control group, the level of ROS in lung tissues was significantly increased in the PQ group.5. Compared with normal control group, the protein expression of Sirtl in lung tissues was significantly decreased in the PQ group.6. Compared with normal control group, the level of TNF-α and IL-6in BALF of mice was significantly increased in the PQ group.The results of the second part:1. Compared with PQ group, the destruction of alveolar structure and collagen deposition were attenuated by pterostilbene (40mg/kg).2. Compared with PQ group, the total cell numbers and total protein concentration in BALF was significantly decreased by pterostilbene (40mg/kg).3. Compared with paraquat group, the cell apoptosis ratio and Caspase-3activity in lung tissues was significantly decreased by pterostilbene (40mg/kg).4. Compared with PQ group, the level of ROS in lung tissues was significantly increased by pterostilbene (40mg/kg).5. Compared with PQ group, the protein expression Sirtl in lung tissues was significantly increased by pterostilbene (40mg/kg).6. Compared with PQ group, the level of TNF-a and IL-6in BALF was significantly decreased by pterostilbene (40mg/kg). Conclusion The down-regulation of Sirtl expression and ROS generation may play an important role in acute lung injury and pulmonary fibrosis following PQ administration. PTS has protective effects against PQ-induced pulmonary fibrosis by suppressing oxidative stress, up-regulating Sirtl expression and inhibiting inflammation in lung tissues.