Resolvin D1 Alleviates the Lung Ischemia Reperfusion Injury via Complement, Immunoglobulin, TLR4, and Inflammatory Factors in Rats

Rv is a newly found lipid molecule that can reduce the inflammation and protect the tissue structure by restraining the proliferation of neutrophils, macrophages, and other inflammatory cells [19‐21]. In chronic disease such as the inflammatory bowel disease and rheumatoid arthritis, Rv can inhibit humoral immunity and cellular immunity to protect the patients [22, 23]. The animal model of skin infection, gingivitis, peritonitis, and sepsis treated by Rv can relieve the injuries, protect the organs, and increase the survival rates of animals [24]. The research about the protective effect of Rv on the IRI organs, for example, heart and brain, has just been started [15, 25]. However, the effects of RvD1 on LIRI are currently unknown.

The ultrastructure of the lung tissue can finely reflect the pathological changes of the lung injury. In the present study, we observed that the normal lung ultrastructure was damaged by IR, exhibiting swelling pulmonary capillary endothelial cells and mitochondria and shrinking nuclear membrane with a large number of inflammatory cells infiltrating in the alveolar septum and capillaries (Fig. 2b). These abnormalities were also found in the H&E staining sections, which showed the destructive alveoli structure, dilated and congestive capillaries, and inflammatory cells filling the thickened interstitium (Fig. 2a). Moreover, the elevated BALF leukocyte count, BALF neutrophil ratio, W/D, and PPI and the decreased level of oxygenation index were also found in the ischemic reperfusion rats. All these above results are potent evidences of the IRI and pulmonary dysfunctions. Through the treatment of RvD1, less structure changes, neutrophil infiltration, and cell apoptosis; lower PPI; and increased oxygenation index were shown in the IR-RV group, indicating a protective effect of RvD1 on lung tissue.

The occurrence of LIRI has become more frequent in clinical practice during the past decades. In recent years, the research of LIRI has been focused on the following aspects: oxygen radicals and lipid peroxidation [26, 27], excessive inflammatory response caused by neutrophil infiltration [28, 29], body fluid and cellular factors [30], imbalance of intracellular calcium homeostasis [31], and cell apoptosis [32]. Among all the five aspects, the immune reaction might be the most critical factor for the pathogenesis of LIRI. Thrane et al. [6] reported that LIRI was a congenital autoimmune reaction. Under the ischemic condition, antigens were exposed and combined with the immune globulin to form the immune complex and activate the complements [8, 33], which could generate a series of bioactive fragments like C3a and C5a and further promote the inflammatory response [34, 35].

LIRI, characterized by nonspecific alveolar damage, hypoxemia, lung edema, and pulmonary hemorrhage, has a direct impact on the prognosis of many related disease, such as the pneumonectomy and the lung transplantation. IR causes lung injury by a variety of reasons. We found that the levels of IgM and IgG and the complements C1q, C2, C3a, C4, and C5a were increased by LIRI. However, after the use of RvD1, they were down-regulated and the damage of lung tissue was alleviated, suggesting that the protective effect of RvD1 was associated with its inhibiting effect on the immune globulin and complements. Tang et al. [36] also presented that i.v. administration of either AT-RvD1 or p-RvD1 caused significant decreases in the BALF contents of neutrophils, inflammatory cytokines, chemokines, and complement C5a. These results suggest a new approach to blocking of immune complex-induced inflammation and protecting for lung injury. Other studies also demonstrated that the inhibition of immune globulins and complements can alleviate the reperfusion injury of organs [37‐39]. The Cr2−/− [40] and RAG−/− [41] immunodeficient mice showed slight IRI due to their incapability of synthesizing IgM. Besides, the level of IRI was aggravated when the mice were treated with IgM or anti-phospholipid antibodies [42, 43]. In addition, LIRI could activate the complement and produce fragments like C3a and C5a, which are important proinflammatory mediators and chemokines, and could recruit the polymorphonuclear neutrophils (PMNs) to the injury sites. Bless et al. [35] reported that the CINC and MIP-2 as well as the complement activation product C5a were required for lung neutrophil recruitment and full induction of lung injury after hindlimb IR in rats. Inhibition of PMN-mediated inflammation can reduce the LIRI [44]. RvD1 is a tissue inflammation-subsided agonist, and its functions are receptor dependent [20, 45]. After LIRI, we applied RvD1 and found less infiltration of PMN in the lung tissue and decreased level of MPO, BALF leukocyte count, and BALF neutrophil ratio, which may be related to the effect of RvD1 on inhibiting complement activation, reducing sICAM-1 and CINC-1 secretion, and promoting ANXA-l expression.

Recent studies have shown that TLR4 plays a key role in the inflammatory cascade of the LIRI [46, 47]. Less damage was found in TLR4-deficient (TLR4 −/−) mice than in wild-type mice (C57BL/6J) in LIRI [48]. NF-κB is a downstream molecule of the TLR4 cascade, which is also important for the autoimmune regulation. The changes of the variety of pro-inflammatory factors, chemokines, adhesion molecules, and enzymes involved in IRI were dependent on the activation of NF-κB [49].

Uncontrolled inflammation usually exists in LIRI. The cellular and molecular changes are controlled by the huge cytokine network. Studies showed that the transcriptional product of NF-κB was the main inflammatory mediator and cytokine in the inflammatory reaction of LIRI, and NF-κB may be the key for the regulation of inflammatory reaction [50]. Thus, NF-κB can affect the LIRI at the transcriptional level by its effects on the inflammatory mediators. Others also showed that LIRI promoted the release of various inflammatory factors, including TNF-α, IL-1, IL-4, IL-6, IL-8, IL-10, and IL-12 [30, 51, 52]. Sharma et al. [53] considered that alveolar macrophages produced TNF-α when experiencing LIRI, which further promoted epithelial cells to release the chemokine, such as KC, MCP-1 and MIP-2, RANTES, and IL-6, and aggravated the injury through neutrophils recruiting under the stimulation of chemokine. Ito et al. [54] found that LIRI increased the concentrations of TNF-α and CINC-1 of the lung tissue. The CINC-1 of rats, corresponding to the human IL-8 family members in the structure and function, has strong effects on the chemotaxis and activation of the neutrophils [49]. However, ANXA-1 has an opposite effect. In vitro and in vivo experiments confirmed that through inhibiting the neutrophil adhesion, migration, and the production of proinflammatory mediators and superoxide, endogenous or exogenous ANXA-l can promote the phagocytosis and apoptosis of PMN and further play an important role in anti-inflammation [55, 56].

Our results showed that the cytokines levels of IL-1β, IL-6, TNF-α, and sICAM-1; inflammatory factors levels of CINC-1, MCP-1, and ANXA-1; and the mRNA level and protein expression of TLR4 and NF-κBp65 were up-regulated under LIRI. The results suggest that the TLR4/NF-κB pathway was activated with the increased release of inflammatory factors, adhesion molecules, and chemokine. Moreover, it has been found that Rv participated in the regulation of the NF-κB pathway [57, 58] and NF-κB was the important signaling molecule and intermediate link of Rv biological effect [59]. RvD1 could decrease the NF-κB-phosphorylated p65 nuclear translocation and inhibit expression of the cytokines and chemokines (TNF-α, IL-1β, IL-6, CINC-1, MCP-1, etc.) during the inflammatory cascade [60‐63]. The inflammatory reaction is one of the vital features of LIRI, and the results of this study were similar with the above previous studies after using RvD1 [60‐63]. Specifically, the cytokines and adhesion molecule of IL-1β, IL-6, TNF-α, and sICAM-1 were notably reduced and the inflammatory mediators of CINC-1 and MCP-1 were decreased but with increased levels of ANXA-1, indicating that the inflammatory condition and lung damage were distinctly relieved.

Inflammation is associated with an oxidative stress reaction, which has a positive feedback on inflammation itself [64]. The results of this experiment showed that the level of MDA, representing the degree of lipid peroxidation and the attack of oxygen radicals [65], was significantly increased in LIRI. However, the activity of SOD and GSH-PX, indirectly reflecting the body’s ability to remove the oxygen radicals [66], was significantly reduced. The results suggested that the decreased ability of scavenging free radicals is also related to the lung damage after LIRI. RvD1 can relieve the injury induced by oxidative stress [64] and protect against oxidative stress-initiated inflammation [67]. In our study, RvD1 significantly improved the SOD and GSH-Px activity, indicating that the scavenging ability of the oxygen free radical was enhanced. Meanwhile, the reduced MDA level confirmed that RvD1 can reduce the free radical-induced lung tissue damage and help to restore the body’s oxygen/antioxidant balance. LIRI increased the apoptosis of lung cells [32], and anti-oxidative and anti-inflammatory treatment could reduce the IR-induced lung cell apoptosis [68]. Our results showed that RvD1 can significantly reduce the apoptosis of lung tissue after LIRI, which may be associated with the effect of RvD1 on inhibiting the oxidative stress and inflammation.

RvD1 mainly plays a role in the inflammatory process, but it would not participate in the maintenance of the physiological functions. Therefore, this new type of anti-inflammatory drug may not interfere with the body’s normal physiological activity and cause no obvious adverse reaction. However, the unstable property, short half-life and high price, the unknown dosage and the administrating timing, the frequency, and the delivery ways would limit its application. Thus, the development of the stable analogue of RvD1 may eventually achieve the purpose of clinical use. Further studies are needed to investigate the potential harm of RvD1 to the immune protective function caused by the inhibitory effect on the complement system, especially when suffering from the surgery and other injuries. This experiment did not detect the dynamic changes of the complement system during LIRI, and it is still needed further study to explore the effect of complement after LIRI by using gene-deficient or gene knockout animals. In addition, the lung tissue has a dual blood supply system and can directly obtain oxygen by pulmonary ventilation, which makes LIRI different from other organs’ IRI [69]. The reported models and our animal models of LIRI were achieved by blocking and then loosening the pulmonary hilus, which blocked not only the pulmonary artery but also the bronchus and bronchial artery, leading to the difference from the clinical LIRI. Much effort is still needed to improve the animal model of LIRI and make it closer to clinical practice.