Pseudoephedrine + emodin inhibits secretion of pro-inflammatory factors and reduces pulmonary edema
The chemical structures of pseudoephedrine and emodin are shown in Fig. 1A.
In the LPS group, microscopic observation of lung tissues after H&E staining revealed thickened lung interstitium with significant neutrophil infiltration and airway hemorrhage. In comparison, lung injury was significantly ameliorated in the treatment group (Fig. 1B). Levels of inflammation were graded based on the degree of observed histological changes in lung tissues (Fig. 1C).
In the LPS group, serum ELISA results revealed significantly increased serum levels of the pro-inflammatory factors TNF-α and IL-1β compared with the control group (Fig. 1D-E). These levels were significantly suppressed in the treatment group. The levels of the same pro-inflammatory factors in lung tissues exhibited similar trends as those from the serum ELISA results (Fig. 1F-G).
In the LPS group, we also found increased mRNA expression levels of inflammasome NLRP3, which is an important activator of IL-1β. In comparison, such mRNA expression levels were suppressed in the treatment group (Fig. 1H).
In the LPS group, Western blot analysis showed suppressed expression of AQP-1 and AQP-5 proteins in lung tissues. In comparison, the expression of these proteins was significantly increased in the treatment group (Fig. 1I-J).
Finally, reduced lung D/W ratio was found in the LPS group. This, too, was significantly improved after treatment with pseudoephedrine + emodin (Fig. 1K).
Collectively, these results indicate that the combination of pseudoephedrine + emodin is effective in reducing LPS-induced inflammatory injuries and pulmonary edema.
Figure 1. Effect of pseudoephedrine + emodin on histopathological changes in lung tissues (200×), expression of AQP-1 and of AQP 5, and inflammatory cytokines in LPS-induced ALI rats. (A) The chemical structures of pseudoephedrine and emodin. (B) H&E staining. (C) Inflammation scores for histopathological changes in lung tissues after H&E staining. (D) ELISA results for serum levels of TNF-α. (E) ELISA results for serum levels of IL-1β. (F-H) Real-time PCR analysis results for TNF-α, IL-1β, and NLRP3 expression levels in lung tissues. (I-J) Western blot analysis results for AQP-1 and AQP-5 protein expression levels. (K) Pulmonary edema, as reflected by D/W weight ratios and lung weight/body weight ratios.All data are expressed as mean ± S.D. (n = 3).## p < 0.01, ### p < 0.001 vs. control group. * p < 0.05,** p < 0.01, *** p < 0.001 vs. LPS group.
Pseudoephedrine + emodin promotes secretion of immunosuppressive factors
IL-10 is a common inflammatory suppressor and arginase-1 (Arg-1) is a classical marker of the alternatively activated macrophages (M2). ELISA and real-time PCR were used to measure the expression of IL-10 and Arg-1 in serum and in lung tissues (Fig. 2). In the LPS group, results revealed stress-induced increase in IL-10 and Arg-1 expression levels in both serum and lung tissues when compared to the control group. These levels were increased in the treatment group in comparison to the LPS group (Fig. 2A-D).
Figure 2. Effects of Pseudoephedrine + emodin on the expression of IL-10 and Arg-1 in LPS-induced ALI rats. (A) ELISA results for serum levels of IL-10.(B) ELISA results for serum levels of Arg-1. (C-D) IL-10 and Arg-1 lung tissue mRNA expression levels. All data are expressed as mean ± S.D. (n = 3).# p < 0.05,## p < 0.01, ### p < 0.001 vs. control group. ** p < 0.01, *** p < 0.001 vs. LPS group.
Pseudoephedrine + emodin affects febrility in ALI rats
In the control group, rat body temperatures remained within normal range throughout the experiment, with no significant changes occurring within eight hours of saline injection (Fig. 3A-B). In contrast, rats in the LPS group exhibited significant increase in body temperature and TRI, which began as an immediate decrease within one hour after LPS injection, before significantly increasing between one to eight hours after injection. In the treatment group, the body temperatures and TRIs of the rats were significantly lower than those of the LPS group, which indicated the effectiveness of the pseudoephedrine + emodin treatment in alleviating febrility in LPS-induced ALI rats.
Figure 3. Effect of pseudoephedrine + emodin on febrility in LPS-induced ALI rats. (A-B) Changes in body temperatures and TRIs. All data are expressed as mean ± S.D. (n = 5).### p < 0.001 vs. control group. ** p < 0.01 vs. LPS group.
VIP intervention affects pro-inflammatory and anti-inflammatory factor expression in LPS, IL-4-induced alveolar macrophages
Macrophages play an important role in immune response. Therefore, we isolated and cultured alveolar macrophages from rats, used LPS and IL-4 to induce macrophage polarization into M1 and M2 macrophages, respectively, and then studied the expression levels of pro- and anti-inflammatory cytokines after pretreatment with different concentrations of VIP.
Figure 4 shows ELISA and real-time PCR results in measuring he expression levels of target cytokine proteins and mRNAs. In the LPS group, the expression of TNF-α, IL-6, and iNOS proteins and mRNA were significantly increased compared with the control group, while the expression of the anti-inflammatory IL-10, Arg-1, and Ym-1 proteins and mRNAs were decreased; macrophage polarization towards M1 was significant. In the IL-4-induced group, the levels of pro-inflammatory-related factors were only slightly increased compared with the control group, which we hypothesize to be stress changes induced by IL-4. There were significant increases in the expression of IL-10, Arg-1, and Ym-1 proteins and mRNAs, and macrophage polarization towards M2 was significant. Increased VIP dose concentration resulted in a decrease in the expression of the pro-inflammatory TNF-α, IL-6, and iNOS proteins and mRNAs, as well as an overall increase in the expression of the anti-inflammatory IL-10, Arg-1, and Ym-1 proteins and mRNAs (Fig. 4).
These results indicate that VIP could effectively inhibit the release of pro-inflammatory factors and increase the expression of anti-inflammatory factors in macrophages.
Figure 4. Effects of VIP on levels of inflammatory and anti-inflammatory cytokines in LPS or IL-4 induced alveolar macrophages. Macrophages were pretreated with VIP (10− 6 ,10− 7, 10− 8 mol/L) for 24 hours, followed by LPS (200ng/mL) or IL-4 (40ng/mL) stimulation for 12 hours. Cells were collected and the levels of TNF-α (A), IL-6 (B), IL-10 (C), Arg-1 (D) were determined using ELISA. (E-J) TNF-α, IL-6, iNOS, IL-10, Arg-1, Ym-1 mRNA expression was determined using Real-time PCR analysis. All data are expressed as mean ± S.D. (n = 3). # p < 0.05, ## p < 0.01,### p < 0.001 vs. control group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS group. +p < 0.05, ++p < 0.01, +++p < 0.001 vs. IL-4 group.
VIP intervention affects alveolar macrophage polarization towards M1 and M2
It is widely known that CD80 and CD206 are surface markers of M1, M2 macrophages, respectively. We used LPS and IL-4 to induce the polarization of VIP-pretreated alveolar macrophages, followed by immunofluorescence staining for CD86, IL-6 and CD206, IL-10, respectively.
Results are shown in Fig. 5. High CD86 expression was clearly observed in alveolar macrophages after LPS induction. In contrast, M1 polarization was significantly suppressed in cells that were pretreated with VIP. Correspondingly, elevated CD206 expression was observed after IL-4 induction, and VIP pretreatment increased polarization towards M2 (Fig. 5A-G). PCR results showed similar results: compared with LPS and IL-4-groups, VIP pretreatment groups exhibited decreased CD86 mRNA expression and elevated CD206 mRNA expression (Fig. 5H-I).
These results indicate that VIP, as an immunoreactive neuropeptide, has a bidirectional regulatory effect of inhibiting M1 polarization and promoting M2 polarization in alveolar macrophages.
Figure 5. VIP inhibited macrophage polarization towards M1 and promoted polarization towards M2 induced by LPS or IL-4-induced. (A-G) Immunofluorescence shows the expression levels of the M1 macrophage markers CD86 and IL-6, as well as the M2 macrophage markers CD206 and IL-10. (G-H) Real-time PCR analysis shows CD86 and CD206 mRNA expression levels. All data are expressed as mean ± S.D. (n = 3). ## p < 0.01,### p < 0.001 vs. control group. * p < 0.05, ** p < 0.01 vs. LPS group. +p < 0.05, ++p < 0.01, +++p < 0.001 vs. IL-4 group.
Pseudoephedrine + emodin treatment induces VIP/cAMP/PKA signaling pathway and inhibits NF-κB signaling pathway
Previous results have shown that VIP can inhibit M1 polarization and promote M2 polarization in alveolar macrophages, thereby showing anti-inflammatory results. In the present study, we examined the expression of the VIP/cAMP/PKA signaling pathway and the NF-κB signaling pathway in the lung tissues of LPS-induced rats after treatment with pseudoephedrine + emodin.
Results are shown in Fig. 6. Pseudoephedrine + emodin treatment effectively promoted VIP protein and mRNA expressions both in serum and in lung tissues (Fig. 6A-D), elevated cAMP and p-PKA protein expression in lung tissues (Fig. 6C-E), and significantly inhibited NF-κB phosphorylation.
This result indicates that pseudoephedrine + emodin treatment exerts anti-inflammatory effects via the VIP/cAMP/PKA signaling pathway.
Figure 6. Pseudoephedrine + emodin increased VIP/cAMP/PKA pathways and inhibited NF-κB in LPS-induced ALI rats. (A) ELISA results for VIP in serum. (B) Real-time PCR analysis results for lung tissue mRNA expression. (C) Western blot results for protein expression. (D) Western blot results for VIP and CAMP protein expression. (E-F) Western blot results for the phosphorylation of PKA, IκBα and P65. All data are expressed as mean ± S.D. (n = 3).## p < 0.01, ### p < 0.001 vs. control group. * p < 0.05,** p < 0.01, *** p < 0.001 vs. LPS group.
Pseudoephedrine + emodin treatment inhibits M1 polarization and promotes M2 polarization in alveolar macrophages
F4/80 is a macrophage surface marker that is commonly used to reflect macrophage activation status and recruitment levels. To determine the effects of pseudoephedrine + emodin on the polarization of macrophages in LPS-induced rat lung tissues, we performed immunohistochemical analysis of F4/80 on rat lung tissue sections (Fig. 7A-B) and performed immunofluorescence analysis of CD80, IL-1β; F4/80, IL-10 (Fig. 7C-H).
Results indicate that the macrophage surface marker F4/80 was highly expressed in both the LPS group and the treatment group compared with the control group. However, macrophages in the LPS group were mainly M1-polarized and formed an inflammatory environment in the lung tissue, whereas the treatment group showed inhibited M1 polarization and increased M2 polarization, as well as lower expression of CD80 and IL-1β, and higher expression of IL-10, which, taken together, formed an anti-inflammatory environment.
When taken together with all aforementioned results, this indicates that pseudoephedrine + emodin treatment can inhibit M1 polarization and promoted M2 polarization via the VIP/cAMP/PKA signaling pathway, thereby producing anti-inflammatory effects.
Figure 7. Pseudoephedrine + emodin inhibited M1 macrophage and activated M2 macrophage in lung tissues.
(A) Immunohistochemistry shows F4/80 expression levels. (B-C) Immunofluorescence shows CD80, IL-1β, IL-10, and F4/80 expression levels. All data are expressed as mean ± S.D. (n = 3).## p < 0.01, ### p < 0.001 vs. control group. *** p < 0.001 vs. LPS group.