Our experiments showed that LPS treatment resulted in severe microstructure damage, oedema and elevated lung tissue permeability, leading to respiratory and circulatory disorders as previously reported [10, 12, 15]. We demonstrated for the first time that prophylactic treatment using SSY prevented respiratory and circulatory functions from damage by LPS through alleviating pathological changes of the lungs in ALI rats. The underlying mechanisms possibly involved anti-inflammation via inhibition of NF-κB signalling pathway and antioxidant stress via activation of the Keap1-Nrf2-ARE signalling pathway, which seemed more effective at a high dose.
ALI triggers excessive inflammation and uncontrolled oxidative stress, producing several pro-inflammatory cytokines and free radicals, but also promotes anti-inflammatory and anti-oxidative responses, possibly acting as compensatory mechanisms to defend against these harmful reactions [10, 16, 17]. Consistent with other studies [16, 17], we found that LPS exposure resulted in high-level inflammatory infiltration and oxidative stress and led to elevated levels of anti-inflammatory and anti-oxidative factors. However, the compensating mechanisms are often insufficient and ALI exacerbations are common. If inadequately treated, it causes gradual dysfunction of extrapulmonary organs (such as heart, liver and brain) and subsequently causes multiple organ failure and death [18, 19].
Most Chinese herbs present in SSY, including Panax ginseng [20], Folium perillae [21], Pueraria lobata [22], Poria cocos [23], Fructus aurantii [24], Platycodon grandiflorum [25], pericarpium citri reticulatae [26], Glycyrrhiza uralensis fisch [27], Costus root [28], Zingiber Officinale Radix [29] and Zizyphi fructus [30], have shown anti-inflammatory and/or anti-oxidative effects. Additionally, Peucedani radix, another component of SSY, exhibits a wide spectrum of pharmacological activities including anti-infective, anti-tussive, anti-asthmatic and expectorant effects, which help relieve symptoms and improve ALI prognosis [31]. In addition, some components of SSY alleviate multiple organ dysfunction triggered by ALI. For example, Pinellia ternate, possessing sedative, hypnotic and anticonvulsant effects, can relieve neuropsychiatric symptoms [32], and Panax ginseng, as well as Glycyrrhiza uralensis fisch, enjoys popularity for their hepatoprotective effects [20, 33]. Based on these pharmacological characteristics, it is reasonable to speculate that the mechanisms resulting in ALI attenuation by SSY involve anti-inflammation, anti-oxidation and organ protection, as our experimental results suggest. To compare different doses of SSY treatment, we used low and high SSY doses for intervention after LPS injection. Both doses could restore lung tissue impairment, but high dose had a more pronounced curative effect, suggesting a potential dose-response relationship between SSY and its therapeutic effects. The characteristic early lesions in ALI include mostly tissue injury (rather than necrocytosis) and mild-to-moderate (reversible) organ function damage [34, 35]. If left to progress, ALI results in tissue necrocytosis, causing serious and irreversible organ dysfunctions that are difficult to reverse medically [34, 35]; thus, we preventively treated rats in the early stage of the ALI instead of those with middle or advanced stages of the disease.
The inactive form of NF-κB, a nuclear transcription factor, is located in the cytoplasm and bound by a variety of inhibitory proteins, the NF-κB inhibitors (IkBs) family. Upon exposure to various stimuli, the IkB kinase complex (IKK) is activated into phospho-IKK, which further phosphorylates IkBs and leads to its ubiquitination and degradation. Without inhibition of IkBs, NF-κB is phosphorylated and translocates to the nucleus, promoting transcription of inflammation-related genes [13]. Our results are consistent with those of studies showing that LPS stimulates phosphorylation of IKKβ, IkBα and p65 and promotes expression of pro-inflammatory factors, indicating NF-κB signalling pathway activation [10, 12, 15]. Our results suggest that SSY suppresses this signalling pathway markedly, especially at a high dose. Among the medicinal herbal concoctions based on SSY, some have been reported to promote IL-10 expression [24, 36]. For example, the mechanism that Fructus aurantii exhibited anti-inflammatory effects in LPS-treated mice was partially through IL-10 expression stimulation [24]. Overall, the LPS-induced high level of IL-10 [10, 16, 17, 24], which was further increased by SSY treatment in our study, suggests an amplification of the anti-inflammatory reaction, but the molecular mechanism involved remains to be elucidated.
Numerous antioxidative metabolites such as NQO1, GCLC and HO-1 are produced as a result of the body’s antioxidant system being activated by ALI to protect against oxidative stress damage. Nrf2, which is kept in the cytoplasm by Keap1 under unstressed conditions, is a transcription factor that regulates expression of antioxidant proteins such as NQO1, GCLC and HO-1 by travelling to nucleus where it binds to antioxidant response elements (AREs) and initiates transcription of antioxidant genes [14]. Our results agree with those of published studies showing that LPS leads to increased production of oxidative metabolites, activating the Keap1-Nrf2-ARE pathway and increasing antioxidant activity [14, 16]. An innovative finding is that prophylactic treatment by SSY further increases antioxidant activity in LPS-treated rats and reduces oxidative stress levels; our evidence suggests this occurs by SSY’s activation of the Nrf2 signalling pathway in a possible dose-dependent manner.
Our results were in accordance with previous studies that inhibition of both inflammation and oxidative stress could attenuate LPS-induced ALI effectively [37, 38]. The molecular mechanisms were involved in NF-κB and Nrf2 signaling pathway, respectively [37, 38]. Besides, their synergistic effect in attenuation of ALI has also been reported [39, 40]. Our study indicates SSY may be worthy of further investigation into a therapeutic agent for ALI.
The major limitation of this study is that rodent model can not fully mimic human disease. Although the results of current study are encouraging, the clinical significance remains to be further investigated. SSY has been used in China for thousands of years. Nevertheless, many questions about its effects are still unclear, especially regarding pharmacological properties. Further research is needed to elucidate pharmacological activities and side effects of the components in the herbal formula.
In summary, preventive use of low- and high-dose SSY treatment stabilised vital signs and attenuated lung histopathology in LPS-induced ALI in rats, and high dose tended to be more effective. The main acting mechanisms included anti-inflammation and antioxidant stress mediated by inhibition of NF-κB signalling pathway and activation of Nrf2 signalling pathway, respectively.