European Review for Medical and Pharmacological Sciences 2020; 24: 461-468 Effects of nalmefene hydrochloride on TLR4 signaling pathway in rats with lung ischemia-reperfusion injury N.-L. ZHANG1, H.-Q. WANG2, J. YANG1, P. YANG3, P. KANG4, T. ZHAO1 1Department of Anesthesiology, People’s Hospital of Rizhao, Rizhao, P.R. China 2Operation Room, People’s Hospital of Rizhao, Rizhao, P.R. China 3Administrative District Clinic, The People’s Hospital of Zhangqiu Area, Jinan, P.R. China 4Rehabilitation Department, The People’s Hospital of Zhangqiu Area, Jinan, P.R. China Abstract. – OBJECTIVE: To investigate the ef- Introduction fect of nalmefene hydrochloride on TLR4 signal- ing pathway in rats with lung ischemia-reperfu- Ischemia-reperfusion is a phenomenon that sion injury. often occurs during major operations and or- MATERIALS AND METHODS: Altogether 64 pure inbred male SD rats were divided into gan transplantation. During this period, in the groups A, B, C, and D according to the principle reperfusion process, the tissue of specific organs of body weight similarity, with 24 rats in each will aggravate the tissue damage to specific or- group. Four groups of rats were respectively gans during reoxygenation, which is considered twisted on the left testis to establish unilateral to be more harmful than ischemia itself1. Isch- testicular torsion rats. Group A was the control emia-reperfusion injury of lung is related to lung group, treated with normal saline, group B was transplantation, extracorporeal circulation, pul- the nalmefene hydrochloride high-dose group, treated with 20 μg/kg of nalmefene hydrochlo- monary embolism extirpation, and pneumonec- ride, group C was the nalmefene hydrochloride tomy, which may lead to pulmonary dysfunction low-dose group, treated with 10 μg/kg of nalme- and serious damage. Among its pathogenesis, ox- fene hydrochloride, and group D was the sham ygen free radicals, inflammatory mediators, and operation group. Lung tissue was collected 60 neutrophils play an important role2. As the mech- h later. Western blotting was used to detect the anisms involved in lung ischemia-reperfusion in- expression levels of HMGB1, TLR4, CD14, and NF-κB protein, qPCR was used to detect the jury are complex and interrelated, showing one of mRNA expression level, and enzyme-linked im- the clear mechanisms can help prevent potential munosorbent assay (ELISA) was used to detect serious complications3. Toll-like receptor (TLR), a the expression levels of inflammatory factors pattern recognition receptor that provides the first IL-17, IL-6, and ICAM-1. line of defense against pathogens such as bacte- RESULTS: The expression levels of HMGB1, ria and viruses, is the initial site for activation of TLR4, CD14, NF-κB protein, mRNA, IL-17, IL-6, inflammatory signals in the lung during ischemia and ICAM-1 in group A were significantly high- 4 er than those in groups B, C, and D (p<0.05), and reperfusion . TLR4 plays a key role in the in- while were significantly lower in group D than in flammatory cascade of lung ischemia-reperfusion groups B and C (p<0.05), and were significantly injury. The downstream molecular nuclear fac- lower in group B than in group C (p<0.05). tor-κB (NF-κB) of the cascade is also important CONCLUSIONS: Nalmefene hydrochloride can for autoimmune regulation. The changes of var- effectively inhibit the signal pathway of TLR4, ious proinflammatory factors, chemokines, ad- and can effectively reduce the injury caused by lung ischemia-reperfusion. The large dose is hesion molecules, and enzymes involved in lung closely related to the good effect, which is wor- ischemia-reperfusion injury depend on the activa- thy of promotion. tion of NF-κB5-7. Activation of TLR4 leads to nu- clear translocation of NF-κB, which in turn leads Key Words: to an increase in the expression level of various Lung ischemia-reperfusion, Nalmefene hydrochlo- inflammatory factors, resulting in injury8,9. High ride, TLR4, CD14, NF-κB, Signal pathway. mobility group protein B1 (HMGB1) participates Corresponding Author: Tao Zhao, MD; e-mail: [email protected] 461 N.-L. Zhang, H.-Q. Wang, J. Yang, P. Yang, P. Kang, T. Zhao in many inflammatory reactions in the body and collected for examination14. Group B was a large can combine with TLR4 to promote the activation dose group of nalmefene hydrochloride. Nalme- of NF-κB, which is an important pathway leading fene hydrochloride (trade name: Jinmeifen, Yuxi to the activation of inflammatory10. However, the Pharmaceutical Co., Ltd., Lingbao, Henan prov- receptor CD14 of lipopolysaccharide can cause ince, China, specification: 1 ML: 0.1 mg, approval activation of TLR4, thus causing inflammatory number: Chinese medicine standard H20080805) reaction11. was injected intravenously 10 min before the left Drug therapy has always been a preferred pulmonary hilum was blocked. The treatment in method to regulate the signal pathway of TLR4 and other places was the same as that in Group A. reduce the injury caused by lung ischemia-reper- Group C was a small dose group of nalmefene hy- fusion. However, studies have found that opioid drochloride. A total of 20 μg/kg of namefene hy- peptides can interact with receptors on the surface drochloride (10 μg/kg) was injected intravenously of pulmonary capillary endothelial cells and play into the tail before the left lung was blocked for 10 an important role in acute lung injury12. However, min. The treatment in other places was the same some studies have shown that nalmefene hydro- as that in Group A15. Group D was a sham op- chloride, a new generation opioid receptor antag- eration group. The left hilar was dissociated by onist, has longer acting time, stronger membrane thoracotomy without clamping. At the same time, permeability, higher bioavailability, and less ad- the tail vein of each group was injected with equal verse reactions, and can effectively reduce lung volume of 0.9% sodium chloride solution. After ischemia-reperfusion injury13, so it is commonly 60 h, rats were executed, and lung tissue was ex- used in lung injury reperfusion. However, this re- tracted. search focused on mechanism analysis, and ana- lyzed the influence of nalmefene hydrochloride on Detecting of Protein Level TLR4 signal pathway in lung ischemia-reperfusion by Western Blotting injury through the rat model. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of the same specimen was taken as an internal reference, the animals of each ex- Materials and Methods perimental group (n=6) were executed 24 h after modeling, and the lung tissues of the extracted Selection of Experimental Animals mice were placed in a refrigerator at –80°C for Sixty-four healthy male SD rats were selected, later use. Western blotting was used to detect the with SPF grade and similar age, weight, and body expressions of HMGB1, TLR4, CD14, and NF- length. The rats were from Hunan SJA Labora- κB proteins. HMGB1/GAPDH, TLR4/GAPDH, tory Animal Co., Ltd. The animal manufactur- and NF-κB/GAPDH represent the relative ex- ing license was SCXK (Hunan) 2019-0003. The pression level. laboratory feeding environment was as follows: 12 h of light, 45%-60% of relative humidity, and Detection of MRNA Expression of TLR4, 23-25°C. This study was approved by the Ethics NF-κB, HMGB1, CD14 by QPCR Committee. Extracting of total RNA Establishment of Animal Model About 50 mg of tissue was put into 1.5 ml of Sixty-four rats were randomly divided into RNAse-free centrifuge tube, 0.5 ml of TRIzol groups A, B, C, and D with 16 rats in each group was added. After being ground to homogenate by by random number table method. Group A was a homogenizer, 0.5 ml of TRIzol was added for the control group. The left porta of lung was dis- placing, and the whole process was about 0.5 h. A sociated after thoracotomy, and the occlusion was total of 200 μl chloroform was added to every 1 removed after 45 min of clamping. The swelling ml TRIzol. After rapid shaking and mixing for 30 of lung tissue was observed after 5 min. Color s, the mixture was placed on ice for 5 min. Then, recovery showed that the lung ischemia-reperfu- it was centrifuged at 4°C for 15 min at 12000 r/ sion model was successfully established. Reper- min. About 400-600 μl of supernatant was trans- fusion was continued for 2 h. Blood was collected ferred to a new centrifuge tube with a pipette gun, through the right common carotid artery. Blood then 500 μl/1 ml of TRIzol isopropyl alcohol was samples were collected and then the rats were ex- added, covered, reversed repeatedly, mixed even- ecuted, the upper lobe tissue of the left lung was ly, placed for 10 min, put into a centrifuge, and 462 Nalmefene hydrochloride in TLR4 signaling pathway in rats with lung ischemia-reperfusion injury centrifuged at 4°C for 10 min at 12000 r/min. The han Herostart Biotechnology Co., Ltd., Wuhan, supernatant was discarded, isopropyl alcohol was Hubei, China), IL-6 ELISA test kit (Shanghai absorbed. Then, 1 ml of 75% ethanol was added, Tongwei Industry Co., Ltd., Shanghai, China), and the mixture was thoroughly mixed. The RNA and ICAM-1 test kit (Shanghai Kalang Biotech- was washed after centrifuging at 4°C for 5 min nology Co., Ltd., Shanghai, China). About 100 at 10000 r/min. The supernatant was discarded, L of standard solution, sample to be tested, and dried naturally for 5-10 min, and 20 μl of DEPC negative and positive control solution were ab- water was added to fully dissolve the total RNA. sorbed into the reaction well. A total of 100 L of biological reaction antibody solution was added, qPCR covered with film, mixed, and placed for 40 min.