The α2AR/Caveolin‐1/p38MAPK/NF‐κB axis explains dexmedetomidine protection against lung injury following intestinal ischaemia‐reperfusion

Abstract Intestinal ischaemia‐reperfusion (I/R) injury can result in acute lung injury due to ischaemia and hypoxia. Dexmedetomidine (Dex), a highly selective alpha2‐noradrenergic receptor (α2AR) agonist used in anaesthesia, is reported to regulate inflammation in organs. This study aimed to investigate the role and mechanism of Dex in lung injury caused by intestinal I/R. After establishing a rat model of intestinal I/R, we measured the wet‐to‐dry specific gravity of rat lungs upon treatments with Dex, SB239063 and the α2AR antagonist Atipamezole. Moreover, injury scoring and histopathological studies of lung tissues were performed, followed by ELISA detection on tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐1β and IL‐6 expression. Correlation of Caveolin‐1 (Cav‐1) protein expression with p38, p‐p38, p‐p65 and p65 in rat lung tissues was analysed, and the degree of cell apoptosis in lung tissues after intestinal I/R injury was detected by TUNEL assay. The lung injury induced by intestinal I/R was a dynamic process. Moreover, Dex had protective effects against lung injury by mediating the expression of Cal‐1 and α2A‐AR. Specifically, Dex promoted Cav‐1 expression via α2A‐AR activation and mitigated intestinal I/R‐induced lung injury, even in the presence of Atipamezole. The protective effect of Dex on intestinal I/R‐induced lung injury was also closely related to α2A‐AR/p38 mitogen‐activated protein kinases/nuclear factor‐kappaB (MAPK/NF‐κB) pathway. Dex can alleviate pulmonary inflammation after in intestinal I/R by promoting Cav‐1 to inhibit the activation of p38 and NF‐κB. In conclusion, Dex can reduce pulmonary inflammatory response even after receiving threats from both intestinal I/R injury and Atipamezole.


| INTRODUC TI ON
Intestinal ischaemia/reperfusion (I/R) injury is a common organ injury in surgical practice in the context of body ischaemia and hypoxia. Various pathological mechanisms can lead to the occurrence of intestinal I/R injury, including intestinal obstruction, severe trauma, intestinal transplantation, intestinal torsion, shock and thrombosis. 1 The pathological mechanism of I/R involves various factors including oxidative stress caused by oxygen free radicals and the role of inflammatory cells, calcium overload and cell apoptosis. 2 is a diffuse inflammatory pulmonary damage caused by excessive reactive oxygen species (ROS), which disrupt normal physiological structures and functions. 5 The main pathogenesis of ALI is an inflammatory response mediated by various inflammatory cells cytokines released, 6,7 which can perturb mitochondrial metabolism and fission. 8 When ALI occurs, the destruction of vascular endothelial cells and alveolar epithelial cells caused by inflammation may imperil normal respiratory function of the lungs, thus reducing the cellular oxygen partial pressure of the lung tissues, causing the body to experience hypoxia. 9 In recent years, intestinal I/R-related lung injury has received extensive attention as a clinically relevant research topic.
Dexmedetomidine (Dex), a highly selective alpha2-noradrenergic receptor (α2AR) agonist, is reported to have certain anti-inflammatory and antioxidant effects, which can exert important protective effects in various organs and tissues. 10,11 Moreover, Dex can upregulate the expression of Caveolin-1 (Cav-1) in the lung tissues of the septic rat. 12 Interestingly, Cav-1 can suppress LPS-induced phosphorylation of p38 mitogen-activated protein kinases (MAPKs) and activation of the nuclear factor-kappaB (NF-κB) pathway, thus playing a protective role against lung injury. 13,14 However, the exact mechanism of the relationship between DEX and the p38MAPK/NF-κB pathway in intestinal I/R-induced lung injury has not been demonstrated. 13 Based on the above research, we proposed that Dex may alleviate lung injury via regulating the α2AR/Cav-1/p38MAPK/NF-κB signalling axis. To test our hypothesis, we established a rat model of intestinal I/R injury to demonstrate the effect of DEX treatment on intestinal I/R-induced lung injury mediated by the α2AR/Cav-1/ p38MAPK/NF-κB axis. The right middle lobes of the lungs were used to determine the wet/dry weight ratio of the lung. The right lower lobes of the lung tissues were crushed, dissolved and separated for Western blot analysis. The remaining lung tissue fluid was put immediately into liquid nitrogen for cryopreservation.

| Animal model establishment of intestinal I/R injury
The rats either received no SMA occlusion procedures (intestinal I/R injury) as sham group or received intestinal I/R injury as experimental groups in four groups: intestinal I/R alone and intestinal I/R injury with reperfusion followed by survival for 6,12,24 or 48 hours. that were injected separately with four different adenoviruses including adenoviruses-short hairpin RNA (Ad-sh)-Cav-1, Ad-sh-negative control (NC), Ad-overexpression (oe)-Cav-1 and Ad-oe-NC, all purchased from Shanghai GeneChem Co., Ltd., at four days before intestinal I/R application. The rats were injected intraperitoneally with 0.3 mL adenovirus at a dose of 1 × 10 9 plaqueforming units (PFU) 4 days before intestinal I/R modelling. The final two experimental groups were treated with both Dex (30 minutes before intestinal I/R) and Ad-sh-Cav-1 (four days before intestinal I/R), or Dex and Ad-sh-NC. The survival number of rats in each group was different, with details shown in Table S1. Four-to-five rats from each group were collected for follow-up experiments. Another 20 rats following intestinal I/R modelling were euthanized at 6, 12, 24 and 48 hours after modelling, and their lung tissues were collected, with 3-5 rats in each group.

| Wet-to-dry lung weight ratio
The extra lung tissues were carefully removed after harvesting the right middle lobe of the lung. The right middle lobe was then rinsed with physiological saline, blotted on filter paper and weighed when wet. The lobe was then dried at 60°C for 48 hours, and the residuum was also weighed. The ratio of wet to dry weight and lung water content were calculated by using the equation [(lung wet weight-lung dry weight)/lung wet weight]. 16

| Haematoxylin-eosin (H&E) staining
The rat intestines and lungs were collected, fixed and embedded in paraffin. The samples were cut into 4μm-thick slices, dewaxed with xylene, and with toluene for 5 minutes, and rehydrated with 100% ethanol for 2 minutes, 95% ethanol for 1 minute, 80% ethanol for 1 minute, 75% ethanol for 1 minute and distilled water for 2 minutes.
All samples were then stained with haematoxylin for 5 minutes and differentiated with dihydrochloric acid and ethanol for 30 seconds.
The slides were soaked in tap water for 15 minutes (or 50°C warm water for 5 minutes) and then placed into eosin solution for 2 minutes. An additional three steps of routine dehydration, clearing, and neutral resin sealing were performed. To remove all traces of water, samples were rinsed in several alcohol baths to complete the clearing process: 95% ethanol for 1 minute, 95% ethanol for 1 minute, 100% ethanol for 1 minute, 100% ethanol for 1 minute, toluene carbonic acid (3:1) for 1 minute, toluene for 1 minute, and xylene for 1 minute. At last, samples were mounted and evaluated via an inverted microscope (Olympus Corporation).

| Evaluation of intestinal and lung injuries
The five-point Chiu score was used to evaluate the degree of the

| Enzyme-linked immunosorbent assay (ELISA)
Interleukin (IL)-6 ELISA kit (PI328, Beyotime, Shanghai, China) was used to measure the IL-6 level of the lung homogenates. The optical density (OD) values of the testing samples were measured, and the level of IL-6 in the sample was calculated from the standard curve generated by the standard samples offered within the ELISA kit. Tumour necrosis factorα (TNFα) (PT516, Beyotime) and IL-1β (PI303, Beyotime) kit testing was also conducted in this experiment. Furthermore, the levels of intestinal mucosal injury markers (i-FABP) and diaminoxidase (DAO) in portal vein blood were determined by an ELISA kit supplied by Wuhan USCN Business, according to the manufacturer's instructions.

| Terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labelling (TUNEL) staining
Apoptosis of hepatocytes was determined using the TUNEL staining kit (G3250, Promega) according to the manufacturer's instructions.
All samples were then photographed and observed under a microscope (BX51, Olympus). 18

| Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
The total RNA was extracted from tissues and cells with TRIzol reagents (15596026, Invitrogen Inc.) and then reverse transcribed into complementary DNA (cDNA) using the Script One-Step RT-PCR kit (Takara Bio Inc). RT-qPCR was then conducted using SYBR Premix EX Taq kit (RR420A, Takara) on an ABI 7500 instrument (Applied Biosystems). All investigations involved at least 3 wells, each repeated in triplicate. The primer sequences are shown in Table S2.
The fold changes were calculated using relative quantification (the 2 -ΔΔCt method) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as loading control.

| Western blot analysis
The total protein was extracted from tissues, and the protein concentration was determined by a bicinchoninic acid (BCA) kit (Thermo

| Statistical analysis
All statistical data were expressed as mean ± standard deviation and processed by SPSS 21.0 statistical software (IBM Corp.). The comparisons between groups were analysed by one-way analysis of variance (ANOVA) with Tukey's post-test. P < .05 indicates that the difference is statistically significant.

| Intestinal I/R induces lung injury in rats
Initially, we constructed an intestinal I/R injury model and observed the changes of inflammation and the degree of lung injury. showed that the lung injury induced by intestinal I/R was a dynamic process, with 6 hours after reperfusion being a critical time point that was selected for the subsequent experiments.

| Dex promotes Cav-1 expression via α 2A -AR activation and mitigates intestinal I/R-induced lung injury
Dex acts as a α 2A -AR agonist with high potency and selectivity, thereby reducing the release of inflammatory factors and cytokines.
Some previous literature has shown that Dex can alleviate intestinal I/R injury. 15 In this study, we further studied the protective effects of Dex against lung injury due to intestinal I/R injury by mediating the expression of Cal-1 and α 2A -AR.
Our results showed that the wet-to-dry lung weight ratio in the I/R group was higher than that of the sham group. On the other hand, the ratio was at the level of the sham group after Dex pretreatment and showed only a minor oedema as opposed to the I/R group ( Figure 2A) (all P < .05). The levels of TNFα, IL-1β and IL-6 were elevated in lung tissues of rats with intestinal I/R injury, along with increased mortality rates (Table S1), but were normalized by the pre-injection of Dex to the control levels along with decreased mortality rates (Table S1) ( Figure (Table S1). We concluded that lung injury in rats can be affected by the promotion of α 2A -AR signalling and consequent inhibition of the expression of p38MAPK

| Dex impairs lung injury induced by intestinal
and NF-κB.

| Dex impairs lung injury induced by intestinal I/R by facilitating Cav-1-dependent p38MAPK/NF-κB pathway inactivation
Down-regulation of Cav-1 can enhance the phosphorylation levels of p38 and the expression of NF-κB and exacerbate LPS-induced acute lung injury. 13 The up-regulation of Cav-1 can regulate the production of pro-inflammatory cytokines TNFα and IL-6 induced by LPS in mouse peritoneal and alveolar macrophages via the p38MAPK pathway. 14 Therefore, in our study, we also analysed the effects of Dex on pulmonary inflammatory injury by promoting Cav-1 to inhibit the activation of p38 and NF-κB in lung injury.
Western blot analysis results found that the sh-Cav-1-2 out of three types of Ad-sh-Cav-1 viruses was the most effective and applicable ( Figure S3A; Figure 4A) were pulmonary interstitial/alveolar haemorrhaging along with oedema and elevated infiltration of inflammatory cells ( Figure S1B; Figure 4F). The results of TUNEL assay indicated that the Ad-oe-Cav-1-2 group had restrained cell death but the Ad-sh-Cav-1-2 group showed aggravation of the apoptotic processes after intestinal I/R injury ( Figure S2B; Figure 4G). Additionally, relative to the I/R + oe-NC group, the mortality rate of the I/R + sh-Cav-1 group increased while that of the I/R+ oe-Cav-1 group decreased (Table S1). In summary, Ad-oe-Cav Meantime, haemorrhage and oedema were present both in the pulmonary interstitium and alveoli, with excessive infiltration of immune cell ( Figure S1CC; Figure 4L) (all P < .05). TUNEL staining showed that the number of apoptotic cells in Ad-sh-Cav-1-2 treated rats was higher than that in Ad-sh-NC treated rats ( Figure S2C; Figure 4M) (all P < .05). Furthermore, the mortality rate was much higher in the I/R+ Dex + sh-Cav-1 group than that in the I/R+ Dex + sh-NC group (Table S1). These results suggested that Dex could alleviate pulmonary inflammation by promoting Cav-1 to inhibit the activation of p38 and NF-κB in intestinal I/Rinitiated lung injury.  Figure S2D; Figure 5F) (all P < .05), with the decreased mortality rate observed (Table S1). In conclusion, Dex reduced pulmonary inflammatory response even in the face of intestinal I/R injury and Atipamezole treatment.

| D ISCUSS I ON
The intestine is one of the most sensitive organs to ischaemia.
Intestinal I/R can not only cause damage to the intestine itself, but also cause damage to many remote organs, even leading to multiple Cav-1 p38 P α2AR/Cav-1/p38MAPK/NF-κB pathway to play a protective role in intestinal I/R lung injury model.
Although we have established that Dex can inhibit the inflammatory response by regulating the p38MAPK/NF-κB pathway during intestinal I/R lung injury, we have not investigated other potentially relevant pathways. For example, an excessive oxidative stress response is one of the important mechanisms in intestinal I/R lung injury, 26 but we did not yet explore whether Dex has any effect on oxidative stress response in this process. Besides, in this work, we mainly focused on the effects of intestinal I/R injury on lung inflammation and apoptosis, but did not pay attention to the changes of ROS due to the lack of sample size, which is also the limitation of our study. In future studies, we will pay more attention to this issue.

| CON CLUS ION
In conclusion, Dex can up-regulate the expression of Cav-1 by promoting α2AR activation, thereby inhibiting the activation of p38 and NF-κB in lung injury caused by intestinal I/R and alleviating lung inflammation injury.

ACK N OWLED G EM ENTS
We would like to acknowledge the helpful comments on this paper received from our reviewers.

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests. Formal analysis (equal); Methodology (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.