Isoacteoside improved the survival rate and histopathological damage in septic ALI in mice.
First, we performed the Kaplan-Meier survival analysis in all experimental groups, and the results showed that the higher dose of ISO (100 mg/kg/day, daily) could significantly improve the mouse 72 h survival rate under CLP surgery. Low doses (10 and 50 mg/kg/ day, daily) had no obvious treatment effect (Fig. 1A). Then, in the second experiment, the lung W/D ratio, which reflects pulmonary edema, was calculated in all groups. An increased lung W/D ratio was observed in the ALI group, which could be reduced by using a higher dose of ISO (Fig. 1B). The histopathological examination showed that hemorrhage, edema, necrosis, alveolar damage, increased thickness of the alveolar wall, infiltration of inflammatory cells and pulmonary consolidation were the classical features that were the easiest to observe in the ALI group. A higher dose of ISO could partially counter the development of these features, while some edema and inflammatory infiltration still exited (Fig. 1C). We also found that the ALI + ISO 100 group had lower scores than the ALI, ALI + ISO 10 and ALI + ISO 50 groups (Fig. 1D). Moreover, the alveolar wall thickness, inflammatory cell infiltration and pulmonary MPO were obviously increased in the ALI groups, which could also be reversed by high dose ISO treatment (Fig. 1D, 1E and 1F).
Isoacteoside reduced acid–base disturbance in septic ALI in mice.
Twenty-four hours after the experiment, arterial blood gas analysis was used to explore whether ISO could reduce the acid–base disturbance induced by ALI. A lower pH, decreased HCO3-, PaO2 and increased PaCO2 were detected in the ALI group, while a higher dose of ISO obviously reduced acidosis and improved the pulmonary oxygenation (Fig. 2).
Isoacteoside reduced the systemic cytokine levels in septic ALI in mice.
On the basis of previous results, a higher dose of ISO (100 mg/kg/ day, daily) exhibited an evident protective effect. Therefore, we chose a dose of 100 mg/kg in the following experiment to reveal the potential mechanisms. CLP surgery resulted in severe local and systemic inflammatory reactions. Serum TNF-α, IL-6 and IL-1β levels in all groups were examined by ELISA to detect the severity of systemic inflammation. Significantly lower serum TNF-α, IL-6 and IL-1β levels were detected in the ALI + ISO100 group than in the ALI group (Fig. 3).
Isoacteoside reduced the BALF cytokine levels in septic ALI in mice.
In addition to the systemic inflammatory reaction, severe local inflammation could also be found in the lung, which was reflected by the BALF cytokine levels. BALF TNF-α, IL-6, IL-1β, IL-2, CXCL1, CCL4 and CCL3 levels were examined by ELISA to detect the severity of local inflammation. Significantly increased BALF TNF-α, IL-6, IL-1β, IL-2, CXCL1, CCL4 and CCL3 levels were also detected in the ALI group compared to those in the control group, which could be reduced by ISO treatment (Fig. 4).
Isoacteoside inhibited the mRNA level of cytokines in septic ALI in mice.
The mRNA level of pulmonary TNF-α, IL-6, IL-1β, IL-2, CXCL1, CCL4 and CCL3 was detected by RT-PCR. The results showed that ISO treatment could significantly reduce the transcriptional level of the aforementioned cytokines (Fig. 5A-5F).
Isoacteoside inhibited the TLR4/MyD88/NF-κB p65 signaling pathway in septic ALI in mice.
As the main classical inflammation-related signaling pathway, the TLR4/MyD88/NF-κB pathway was also examined in this study. TLR4, MyD88, p-NF-κB p65, p-IκBα and pIKKαβ expressions were markedly increased in the ALI group, and ISO treatment reversed the increase in protein expression levels (Fig. 6).
Isoacteoside reduced pulmonary oxidative stress in CLP-induced ALI in mice.
Excessive oxidative stress is another key mechanism involved in ALI. We detected the levels of MDA and 8-OHdG, which represented the pro-oxidative stress indexes, and the levels of CAT, SOD, GSH, and GSH-px, which represented the antioxidative stress indexes. The results showed that the MDA and 8-OHdG levels were significantly higher in the ALI group than in the ALI + ISO 100 group (Fig. 7A and 7B). In contrast, ISO treatment significantly increased the CAT, SOD, GSH and GSH-px levels after CLP surgery (Fig. 7C-7F). The results implied that ISO could inhibit pulmonary oxidative stress and improve antioxidative ability.
Isoacteoside inhibited pulmonary endothelial hyperpermeability in septic ALI in mice.
Excessive edema and increased endothelial permeability aggravated ALI. Not only proteins but also inflammatory cells can escape from the blood vessels to the alveoli because of severe vascular leakage. First, we detected the protein and albumin levels in BALF in all groups, and the results showed that the BALF protein and albumin levels were significantly increased after CLP surgery, and these increases could be reduced by ISO treatment (Fig. 8A and 8B). Then, we also tested the total cells, macrophages, neutrophils and MPO in the BALF, which can reflect the leakage of living cells. We found that the total cells, macrophages, neutrophils and MPO levels in BALF were significantly lower in the ALI + ISO 100 group than those in the ALI group (Fig. 8C-8F). Moreover, we tested endothelial junction proteins by western blotting for mechanistic research. The results showed that ISO treatment enhanced the expression levels of ZO-1, claudin-5 and VE-cadherin (Fig. 8G-8H).