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.