LIU Yang, LIU Jie-ting, WANG Ying-bin?

1. Department of Anesthesiology, Lanzhou University Second Hospital, Lanzhou 730030, China

2. The Second Clinical Medical School,Lanzhou University, Lanzhou 730030, China

Keywords:Clemastine fumarate Reperfusion injury Intestinal Acute lung injury

ABSTRACT Objective: To evaluate the protective effect and mechanism of clemastine fumarate (CLE) on acute lung injury (ALI) in intestinal ischemia-reperfusion (I/R) mice.Methods: Twenty-four SPF Balb/c mice were randomly divided into sham operation group (sham group), ischemiareperfusion group (I/R group), and clemastine fumarate pretreatment group (I/R+C group).In the I/R group, an intestinal ischemia-reperfusion model was established (ischemia for 40 minutes, reperfusion for 2 hours). In the I/R+C group, CLE 5 mg/kg was intraperitoneally injected before the operation. Lung tissue morphology was observed and scored by HE staining; and the ratios of wet weight to dry weight (W/D) were recorded. the levels of MDA,SOD, GSH-px, NF-κB and TNF-α in lung tissue of each group were determined by ELISA;Western blot method was used to determine the expression of TLR4 protein in lung tissue.Results: Compared with the Sham group, the I/R group had significantly higher lung tissue injury score and wet/dry ratio (P<0.05), increased lung tissue MDA level (P<0.05), decreased SOD and GSH-px levels (P<0.05), and increased NF-κB and TNF-α levels, the expression of TLR4 protein in lung tissue increased (P<0.05); compared with the I/R group, the lung tissue injury score and wet/dry ratio of the I/R+C group decreased (P<0.05), the level of MDA in lung tissue decreased (P<0.05), the levels of SOD and GSH-px increased (P<0.05), and the levels of NF-κB and TNF-αdecreased (P<0.05), the expression of TLR4 protein in lung tissue decreased (P<0.05).Conclusion: Clemastine fumarate can alleviate acute lung injury after intestinal ischemia-reperfusion in mice, and the mechanism may be related to the inhibition of oxidative stress and inflammatory response in lung tissue.

1. Introduction

Intestinal ischemia-reperfusion (I/R) is a fatal disease with a mortality rate as high as 60% to 80%. Intestinal I/R injury can lead to impaired intestinal mucosal barrier, inducing severe systemic inflammatory response and consequent distal organ damage. The lung is a highly sensitive organ involved in intestinal I/R, and its structure and function are easily affected, causing acute lung injury(ALI)[1]

Clemastine fumarate (CLE), a second-generation histamine 1 receptor (H1R) antagonist, is widely used in the treatment of allergic diseases. Studies have found that H1R-mediated inflammatory response plays an important role in the formation of lung injury, and CLE played an anti-inflammatory role and alleviated lung injury in a model of pulmonary ischemia-reperfusion [2].In clinical studies,the use of CLE before laparotomy in gastrointestinal emergencies has been shown to have a protective effect on the lungs[3]. However,there is currently no definite evidence that CLE plays a protective role in ALI induced by intestinal I/R. Therefore, the mouse intestinal I/R model was used in this experiment to explore the effect and possible mechanism of CLE on lung tissue after the model was established.

2. Materials and methods

2.1 Materials

2.1.1 Animals

24 adult SPF male Balb/c mice were purchased from the Laboratory Animal Center of Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences, weighing 18～25 g. The animals were reared in the animal room of the Cuiying Experimental Platform, the Second Hospital of Lanzhou University, at room temperature of 24～26℃, alternating day and night for 12 hours, and were allowed to eat and drink freely. After a week of adaptation,enter the experimental process. Animal experiments have been approved by the Laboratory Animal Ethics Committee of the Second Hospital of Lanzhou University (batch number: D2022-130).

2.1.2 Reagent clemastine fumarate (batch number

S1847-04, Shanghai Selleck.); MDA ELISA kit (batch number:16203A, Shanghai Sinobestbio.); SOD ELISA kit (batch number:13963A, Shanghai Shanghai Sinobestbio.); GSH-px ELISA kit (batch number: 20963A, Shanghai Sinobestbio.); NF-κB ELISA kit (batch number: 15171B, Shanghai Shanghai Sinobestbio.); TNF-ELISA Kit (batch number: 29312B, Shanghai Shanghai Sinobestbio.);TLR4 antibody (batch number: 66350-1-Ig, Proteintech); dimethyl sulfoxide (batch number: 302A0327, Solarbio); hematoxylin-eosin staining solution (Solarbio) ; paraformaldehyde (Tianjin Guangfu).

2.2 Methods

2.2.1 Grouping and model replication24 Balb/c mice were randomly divided into 4 groups (n=8): sham operation group (Sham group), ischemia-reperfusion group (I/R group) and fumaric acid group Clemastine pretreatment group (I/R+C group). Mice in each group were fasted for 12 hours before surgery and had free access to water. One hour before surgery, CLE 5 mg/kg was intraperitoneally injected in I/R+C group, and 2%DMSO 10 mL/kg was intraperitoneally injected in Sham group and I/R group. I/R group and I/R+C group reference literature to prepare mouse intestinal I/R model [4], ischemia for 40 minutes, reperfusion for 2 hours; Sham group underwent the same operation without clipping. After overdose anesthesia, a "T"-shaped incision was made in the chest with scissors, and bilateral lung tissues were obtained.

2.2.2 Pathological detection of lungTissue 4% formaldehyde was taken to soak the lower lobe lung tissue of the left lung, after dehydration, paraffin embedding,sectioning (5 μm), HE staining, light The pathological changes of lung tissue were observed under microscope. Lung injury was scored according to the degree of pulmonary interstitial edema, intraalveolar inflammatory cell infiltration and intra-alveolar exudation.The criteria are as follows [5]: 0 points: normal, alveolar space area >85%; 1 point, alveolar space area 75%-85%; 2 points: alveolar space area 50%-75%; 3 points, alveolar space area 25% ～50%; 4 points:0%～25% of alveolar cavity area.

2.2.3 Lung wet-dry weight ratioTake the upper lobe of the left lung of the mouse, weigh it, and record the wet weight (W); then dry it in an oven at a constant temperature of 80℃ for 24 hours, dehydrate, weigh, and record the dry weight (D). Calculate the ratio of wet weight to dry weight (W/D).

2.2.4 Lung tissue specimen processing and ELISA detectionFrozen lung tissue was taken, fully crushed lung tissue, centrifuged at low temperature, and the supernatant was collected. The levels of MDA, SOD, GSH-px, NF-κB and TNF-α in the supernatant were detected according to the instructions of the ELISA kit.

2.2.5 Western blot detection of TLR4 protein in lung tissue

Frozen lung tissue was taken, fully crushed lung tissue, lysed in an ice bath, centrifuged at low temperature, and the supernatant was taken for protein concentration determination by BCA method. Protein samples were denatured by boiling, SDS-PAGE electrophoresis, protein transfer, blocked with 5% nonfat milk powder, incubated with PVDF membrane primary antibody overnight (TLR4, 1:1000; β-actin, 1:4000), and incubated with secondary antibody for 1 h, TBST Dip. The PVDF membrane was dripped with ECL developer solution and exposed to light for development. The gray value analysis of protein bands was performed using image J software.

2.3 Statistical analysis

Statistical processing was performed using graphpad prism 9.1.1 statistical software. Measurement data were expressed as mean ± standard deviation (±s), comparison between groups was performed by one-way ANOVA, Ordinary ANOVA test was used for homogeneity of variance, and Brown-Forsythe and Welch ANOVA test was used for heterogeneity of variance. P<0.05 means the difference is statistically significant.

3. results

3.1 The results of pathological changes in lung tissue and the results of W/D ratio

In the Sham group, the lung tissue was intact, the alveolar and interstitial structures were clear, and no obvious abnormality was found. Compared with the Sham group, the lung injury score and the W/D ratio of the I/R group were significantly increased(P<0.05), there was exudation in the alveoli, the alveolar rupture and fusion, and the pulmonary interstitium was significantly thickened, accompanied by a lot of inflammation. Cells aggregated and infiltrated. Compared with the I/R group, the lung injury score and the W/D ratio of the I/R+C group were decreased (P<0.05).(Figure1, Table 1.)

Tab 1 The lung morphology and wet/dry ratio of mice in each group(n=8, ±s)

Tab 1 The lung morphology and wet/dry ratio of mice in each group(n=8, ±s)

*P＜0.05 compare with Sham group;#P＜0.05 compare with I/R group

Group Lung morphology score W/D Sham 0.25±0.45 3.58±0.38 I/R 3.33±0.49* 4.39±0.17*I/R+C 1.50±0.67*# 3.92±0.16*#F 69.81 16.85 P P<0.000 1 P<0.000 1

3.2 Expression of MDA, SOD and GSH-px in mouse lung tissue

Compared with the Sham group, the expression of MDA in the lung tissue of the mice in the I/R group and the I/R+C group was significantly increased, while the expression of SOD and GSH-px was decreased (P<0.05). The expression of MDA in the lung tissue of I/R+C group decreased, while the expression of SOD and GSHpx increased (P<0.05). (Tab.2)

Tab 2 Expression levels of MDA, SOD and GSH-px in lung tissue of mice in each group(n=8, ±s)

Tab 2 Expression levels of MDA, SOD and GSH-px in lung tissue of mice in each group(n=8, ±s)

*P＜0.05 compare with Sham group;#P＜0.05 compare with I/R group

Group MDA (nmol/mL) SOD (ng/mL) GSH-px (pg/mL)Sham 5.37±0.43 28.82±1.11 563.70±35.7 I/R 11.37±0.74* 13.49±1.48* 299.10±15.49*I/R+C 7.40±0.65*# 23.75±1.62*# 354.80±30.29*#F 253.9 235.8 234.3 P P<0.000 1 P<0.000 1 P<0.000 1

3.3 Expression of NF-κB and TNF-α in mouse lung tissue

Compared with the Sham group, the contents of NF-κB and TNF-α in the lungs of the mice in the I/R group and the I/R+C group were significantly increased (P<0.05); compared with the I/R group, the I/R+ The contents of NF-κB and TNF-α in the lungs of mice in group C decreased (P<0.05). (Tab.3)

Tab 3 Expression levels of NF-κB、TNF-α in lung tissue of mice in each group(pg/mL,n=8, ±s)

Tab 3 Expression levels of NF-κB、TNF-α in lung tissue of mice in each group(pg/mL,n=8, ±s)

*P＜0.05 compare with Sham group;#P＜0.05 compare with I/R group

Group NF-κB(pg/mL) TNF- (pg/mL)Sham 592.50±43.33 496.60±46.59 I/R 1204.00±47.79* 836.10±31.67*I/R+C 725.60±87.76*# 556.10±61.23*#F 311.1 188.6 P P<0.000 1 P<0.000 1images/BZ_23_2055_1757_2088_1790.png

3.4 Expression level of TLR4 protein in mouse lung tissue

Compared with the Sham group, the expression of TLR4 protein in the lung tissue of the mice in the I/R group was significantly increased (P<0.05); compared with the I/R group, the expression of TLR4 protein in the lung tissue of the I/R+C group was significantly decreased (P<0.05). <0.05). (Tab.4, Fig.2)

Tab 4 Expression levels of TLR4 in lung tissues of mice(n=4, ±s)

Tab 4 Expression levels of TLR4 in lung tissues of mice(n=4, ±s)

*P＜0.05 compare with Sham group;#P＜0.05 compare with I/R group

Group TLR4/β-actin Sham 0.427±0.043 I/R 0.618±0.062*I/R+C 0.477±0.048#F 14.98 P 0.001 8

4. Discussion

Intestinal I/R injury is one of the most common tissue and organ injuries during surgery, such as abdominal aortic aneurysm surgery,heart-lung bypass surgery, and bowel transplantation[6]. Intestinal I/R injury can generate reactive oxygen species (ROS), cytokines,and chemokines, which in turn activate the immune system, leading to distal organ dysfunction, especially in the lungs, and triggering ALI[7].

The imbalance between the antioxidant system and excessive free radical production in lung injury is an important cause of lipid peroxidation in biofilms and severe cell damage[8]. MDA is one of the main end products of lipid peroxidation caused by ROS,and its elevated level indicates increased oxidative stress in the body; SOD and GSH-px are important components of the body's antioxidant enzyme system, and elevated activity indicates the body's antioxidant capacity. The balance between the two can indirectly reflect the degree of oxidative stress in the body[9]. Studies have found that factors such as MDA and SOD in lung tissue are involved in the occurrence and development of ALI [10]. Excessive oxidative stress is one of the main pathological processes after intestinal I/R. Intestinal I/R local oxygen free radicals increase excessively,spread to the whole body with blood circulation, stimulate chain lipid peroxidation, and act on type II Alveolar epithelial cells, which eventually lead to ALI[5]. Studies have found that in the mouse intestinal I/R model, the degree of pulmonary edema increases,the lung injury is aggravated, the MDA in the lung increases,and the GSH decreases [5]. The mouse intestinal I/R model was established in this experiment, and it was found that the structure of the lung tissue of the mice was significantly damaged, the W/D ratio increased, the alveolar exudation increased, the pulmonary interstitium was thickened, and a large number of inflammatory cells were accumulated. The expression of MDA was significantly increased, and the SOD and GSH-px were significantly decreased,suggesting that ALI and oxidative stress occurred after intestinal I/R in mice.

CLE is an H1R antagonist, and H1R antagonists can reduce oxidative stress and inhibit the production of mitochondrial ROS[11]. It has been reported that the activation of mast cells(MC) exacerbates intestinal I/R-induced oxidative stress and ALI,and MC activation can promote the production of intracellular reactive oxygen species, resulting in a decrease in SOD activity and an increase in MDA levels[12]. In a model of organ ischemiareperfusion, it was confirmed that CLE could inhibit MC activation and degranulation by downregulating H1R[13]. In this study, it was found that after CLE intervention, the pathological damage of lung tissue was alleviated, the W/D ratio decreased, and the expression of MDA in the lung tissue of mice after intestinal I/R was inhibited,and the expressions of SOD and GSH-px were increased, suggesting that CLE may Inhibition of oxidative stress alleviates ALI.

Inflammatory response is another important cause of intestinal I/R leading to ALI. Toll-like receptors (TLRs) are pattern recognition receptors that are mainly involved in the body's innate immune process. TLR4, a member of the TLRs family, can recognize various ligands and activate multiple signaling pathways, including MAPK and NF-κB pathway proteins, and play an important role in regulating inflammatory responses[14]. Studies have shown that TLR4/NF-κB/TNF-α signaling is a key pathway regulating intestinal I/R-induced ALI[15]. The results of this experiment showed that the expression of TLR4 protein in lung tissue was significantly increased after intestinal I/R, and the contents of NF-κB and TNF-α in the lung were up-regulated, suggesting that the TLR4/NF-κB/TNF-α inflammatory pathway plays an important role in ALI . It has been reported that intestinal I/R treatment of TLR4 knockout mice can reduce the infiltration of inflammatory cells in the lung,thereby reducing lung injury, and down-regulating the levels of NFκB and serum TNF-α in the lung[16]. Recent studies have shown that CLE can reduce the inflammatory response after ischemiareperfusion in the lung [17] and myocardial[18], and reduce tissue damage by inhibiting the expression of TLR4 protein. The results of this experiment showed that CLE inhibited the expression of TLR4 protein in lung tissue after intestinal I/R and down-regulated the levels of NF-κB and TNF-α in the lung, suggesting that CLE may reduce the risk of mice by inhibiting the TLR4/NF-κB/TNF-α pathway. Intestinal I/R-induced inflammatory response to ALI.

There are still limitations in this study. First, CLE is an H1R antagonist, and the role of H1R in ALI is not involved in this study.In the follow-up study, gene silencing or knockout technology of histamine receptor can be selected for identification research.Secondly, this study explored the role of oxidative stress and TLR4/NF-κB/TNF-α signaling pathway in ALI after intestinal I/R, but its upstream signaling still needs further study.

In conclusion, CLE can alleviate ALI after intestinal I/R in mice,and the mechanism may be related to the inhibition of oxidative stress and inflammatory response in lung tissue.

Conflict of interest statement: All authors have no conflict of interest.

Contribution of the author: Liu Yang participated in the animal feeding, experimental operation and writing the paper; Liu Jieting guided the experimental operation and data statistical analysis; Wang Yingbin guided the experimental design and thesis revision.