Fig 6. Effects of AS-IV treatment on poly (I: C)-induced lung inflammatory lesions in mice. Mice infected with poly (I: C) were treated with the specified concentration of AS-IV (25, 50 mg/kg /d), and the mice were sacrificed after 6 days of treatment with AS-IV to evaluate the protective effect of AS-IV on mice. (A) H&E staining of lung tissue. (B) Pathological scores of lung tissue in mice of each group. (C) Lung index for mice sacrificed at the day 6. All data are expressed as mean ± SD, (n= 10 per group). * P < 0.05, ** P < 0.01.
4. Discussion
Influenza virus infection has been a great challenge to the global health system, causing a large number of deaths and economic losses worldwide every year [3]. The initial host response to influenza virus invasion is acute inflammation, one which is characterized by the activation of inflammatory cytokines or chemokines, leading to the recruitment of inflammatory cells [27]. A moderate immune response will help clear the virus, while excessive immune stimulation can cause tissue and organ damage, and a large number of inflammatory cytokines will even overflow into the circulatory system, causing a systemic cytokine storm, resulting in multiple organ dysfunction [28,29]. This serious consequence urgently requires the development of drugs to reduce lung damage caused by influenza virus, and the secondary development and application of existing drugs seems to be more advantageous.
Traditional Chinese herbs have been used to prevent and treat viral infections for hundreds of years and are popular around the world due to their good tolerance. AS-IV is a natural compound with multi-target therapeutic properties extracted from Radix Astragali , which has a good antiviral effect on hepatitis B virus [30], Coxsackie virus, [31,32] and dengue virus [33], etc. Its antiviral function has also been widely verified in respiratory diseases [25,34,35]. Although Zhang et al. found that AS-IV reduces the level of IL-1β in influenza virus infection, the specific mechanism of action of AS-IV in influenza virus infection remains unclear. Here, we further investigated the effect of AS-IV on influenza virus infection and its underlying mechanism which may contribute to the application of AS-IV and its analogues in the therapy of severe viral pneumonia.
ROS is a by-product of biological aerobic metabolism and a general term for a class of oxygen-containing and active substances. The balance of oxidative and antioxidant mechanisms is the key to maintaining ROS levels during physiological metabolism. Appropriate levels of intracellular ROS are necessary for signal transduction and apoptosis during cell growth, but excessive accumulation of ROS can cause oxidative stress leading to cell death [36,37]. After infecting the host, the influenza virus reduces and consumes the antioxidant oxidase activity of TAC, GPX, SOD, CAT, and other antioxidant systems, thereby increasing the ROS content in the body, leading to the occurrence of oxidative stress [38-40]. Antioxidant enzymes can terminate free radical chain reactions, and MDA is the end product of free radical chain reactions and is widely used to measure the degree of oxidation deterioration in biological systems [41]. Our results showed that compared with the NC group, influenza virus infection decreased the activities of TAC, SOD, GPX, and CAT in A549 cells, and significantly increased MDA and ROS levels, suggesting that influenza virus infection induced oxidative stress injury of A549 cells. After AS-IV treatment TAC, GPX, SOD, and CAT were significantly increased, while MDA and ROS levels were significantly decreased, suggesting that AS-IV has a strong antioxidant effect. These data indicate that AS-IV also exhibits the ability to inhibit ROS and antioxidants in influenza virus infection. Our subsequent experiments showed that the mRNA and protein levels of NLRP3 were significantly increased in A549 cells after influenza virus infection, and its downstream Caspase-1, IL-18, and IL-1β were also increased. When AS-IV inhibited intracellular ROS levels in a dose-dependent manner, intracellular NLRP3, Caspase-1, IL-18, and IL-1β levels were all reduced in a dose-dependent manner. Our data demonstrate that AS-IV may inhibit influenza virus-induced inflammation mediated by the ROS/NLRP3/Caspase-1 signaling pathway in vitro .
In vivo , we selected poly (I: C) to simulate viral infection in mice. Poly (I: C) is a double-stranded RNA analog that can cause lung inflammation, interstitial edema, bronchiolar epithelial hypertrophy, and lung function changes in mice, and can induce pro-inflammatory factors IL-6, TNF-α, Elevated levels of IL-1β and IL-8 and increased inflammatory cells [42,43]. Our results showed that the lung tissue structure of mice in the poly (I: C) group was severely damaged, and the pathological score and lung index were significantly increased compared with the normal control group. While in the therapy group, AS-IV significantly alleviated the destruction of lung tissue structure and decreased the pathological score and lung index of mice. These results demonstrate that AS-IV can effectively attenuate viral lung injury.