Hemorrhagic shock is a common complication following trauma and surgery, as well as one of the common issues in emergency cases and critical illnesses [1,2]. Hemorrhagic shock can cause extensive organ dysfunction and metabolic disorders. The death rate yielded by trauma-associated hemorrhagic shock is relatively high, and the primary cause of death in this condition is multiple organ dysfunction syndrome (MODS) induced by sharp reduction in the effective circulating volume, tissue hypoperfusion, reperfusion injury, and endotoxin translocation, among others . Additionally, acute kidney injury (AKI) is one of the primary causes of death among patients with MODS. AKI incidence constantly increases, and irreversible reduction in renal function may be subsequently found in some patients who have suffered from AKI, potentially resulting in chronic kidney disease (CKD) or end-stage renal disease (ESRD) and ultimately in reduced survival of patients . AKI is an independent risk factor for CKD and ESRD, as well as death and other diseases related to kidney disorders.
The microenvironment of macrophages in the kidney changes resulting from the release of large amount of inflammatory mediators when ischemic injury occurs in the kidney . Therefore, in addition to active fluid resuscitation therapies at the early stage of hemorrhagic shock, anti-inflammatory treatment measures that block or reduce the production and release of inflammatory mediators can significantly prevent the progress of hemorrhagic shock . The cholinergic antiinflammatory pathway is a vagal-mediated pathway for real-time regulation of inflammatory response. When an inflammatory response occurs in the human body, vagal afferent neurofibers are stimulated by local inflammatory factors to transfer the inflammatory signals to the hypothalamus [7, 8]. Acetylcholine is then released into the inflammatory site through vagal efferent neurofibers to activate nicotinic ACh receptor 7 (nAChRα7), thereby inhibiting the activation of monocyte macrophages and reducing the release of proinflammatory factors, such as TNF-α, and ultimately regulating the inflammatory response [9, 10, 11]. This study performed isolation and culture of macrophages in the kidney to explore the regulatory function of miR-132 in the cholinergic pathway and its effect on the macrophages.
2 Materials and methods
2.1 Culture of macrophages in the kidney
All procedures in the animal experiments were conducted in accordance with the guidelines developed by the National Institutes of Health and approved by the Institutional Animal Care and Use Committee of Shenzhen Peking University - The Hong Kong University of Science and Technology Medical Center (Permit SYXK 2015-2016). 10 male eight-week-old SD rats (220g to 250g) were sacrificed by cervical dislocation and disinfected by soaking in 75% alcohol for 5 minutes. The rats were retrieved and positioned upside down, and the bilateral kidneys were surgically removed under sterile conditions. The kidneys were placed in a flat plate filled with precooled RPMI-1640 (Gibco, NY, USA) culture solution (containing 1% calf serum), and the connective tissues and fats were removed. The kidney tissue was squeezed through steel wire gauze (200 mesh) to obtain single cells, which were centrifuged at 200 ×g for 10 minutes and the supernatant was then extracted. A cell solution containing approximately 1 × 108 cells/ml was prepared using RPMI-1640 culture solution containing 1% calf serum. The single cells obtained were inoculated with DMEM culture solution containing 10% FBS (Gibco, NY, USA) in a cell culture flask and placed in CO2 incubator with 5% volume fraction at 37°C under saturated humidity. The macrophages were purified using an adherent method.
2.1.1 Construction of oxygen–glucose deprivation (OGD) model of renal macrophages
The renal macrophages were inoculated on a 24-well plate at a density of 3.0 × 104 cells/ml with 1 ml of culture medium per well. After 24 hours, the culture solution was replaced with serum-free medium and then cultured for another 24 hours; the original culture solution was discarded. For cells in the normal group, Earle’s fluid was added; for cells in the hypoglycemia and hypoxia groups (10%XB+OGD), Na2S2O4 sugar-free Earle’s fluid (4 mmol/ml) was added after the cells were washed once with sugar-free Earle’s fluid. The duration for induction of injury under hypoglycemia and hypoxia was 1 hour. Earle’s fluid was used to wash the cells once after 1 hour. Complete culture solution (10% FBS+1640) was added into the culture, and then the cells under hypoglycemia and hypoxia were divided into two groups; ACh (10×10−5 mol/L, 10%XB+OGD+ACh) was added in one group, whereas the mixture of ACh (10 ×10−5 mol/l) and galanthamine (Gal) (10 μmol/l) was added into the other group and then cultured for 24 hours (10%XB+OGD+ACh+Gal). All of the s were repeated three times.
2.2 Cell transfection
Renal epithelial cells were inoculated in a 12-well culture plate, resulting in 50–70% cell confluency when transfection was performed. miRNA-132 (RiboBio Co., LTD, Guangzhou, China) was overexpressed, and the control vector (6 μg) was transfected into the cells using Lipofectamine 2000 (Invitrogen, USA). The mixed liquor was removed after 8 hours, and then 2 ml of DMEM was added into the complete culture solution. The cells were observed under a fluorescent microscope at 3–5 days after transfection. After miRNA-132 was stably expressed in renal epithelial cells, the abovementioned renal macrophages under hypoglycemia and hypoxia were inoculated in a 96-well culture plate at a density of 5 × 103 cells/cm2. When the cells were overgrown by 50–60%, the DMEM culture solution containing 10% FBS was placed in a double cell culture system containing renal tubular epithelial cells; this culture system was used for transfection and stable expression of miRNA-132 for 24 hours.
2.3 Enzyme-Linked Immunosorbent Assay (ELISA)
The expression levels of TNF-α, IL-1β, IL-10, TGF-β, IL-4 and IL-6 in cultured cell supernatant were determined using ELISA kit (Senxiong Technology Industrial Co., Ltd., Shanghai, China). The absorbance at 450 nm was detected by a microplate reader, and all the results were standardized using a standard curve.
2.4 Cell viability evaluation (MTT assay)
Approximately 1 × 104 cells were inoculated in a 96-well plate, which was subsequently placed in a 5% CO2 cell incubator at 37°C to culture for 24 hours. Appropriate concentrations of the tested compounds were added into the culture solution under suitable conditions and then cultured for 24 hours. Afterwards 1× MTT (50 μL) was added into each well and incubated at 37°C for 4 hours. The supernatant was removed, 150 μL of DMSO was added into each well, and the optical density at 570 nm in each well was determined by a microplate reader.
2.5 Real-time quantitative PCR (RT-qPCR)
A randomized selection of 6 rat kidney macrophages were performed the relative expression levels of miR-132, AChE, AChE-R and AChE-S mRNA were determined by RT-qPCR. TRIzol kit (Invitrogen, USA) was used to extract the total RNA of the renal macrophages. The absorbance of RNA was tested at 260 and 280 nm, and its expression level and purity were determined using an ultraviolet spectrophotometer. Reverse transcription was performed using PrimeScript RT reagent Kit (TaKaRa Biotechnology Co. Ltd., Shanghai, China), total system 10 μL per reaction. RT-qPCR was completed using SYBR Premix Ex Taq II kit (Applied Biosystems, Foster City, CA, USA), total system 20 μL per reaction. PCR amplification was completed using a one-step quantitative PCR system, and U6 snRNA and β-actin were used as standard internal references for the expression levels of miR-132, AChE, AChE-R and AChE-S mRNA. The 2-ΔΔCt method was used for graphical representation of data, and the difference in the samples was evaluated. The kits mentioned above were used in accordance with the manufacturer’s instructions. The primers for miR-132, AChE, AChE-R and AChE-S U6 snRNA, and β-actin are listed in table 1. All of the experiments were repeated three times.
2.6 Western blot analysis
To test the AChE and control GAPDH protein, we extracted the total protein of renal macrophages using RIPA, and the same amount of protein sample was used for loading. The total proteins were incubated with blocking buffer for 2 hours after electrophoresis, decolorized with TBST at room temperature, and then washed twice on a shaking table for 10 minutes each time after the AChE antibody (Abcam, Cambridge, MA, USA, ab188358) and GAPDH antibody (Santa Cruz, USA, sc-365062) primary antibody was diluted with TBST to 1:500 and 1:1000 prior to incubation at room temperature for 1–2 hours. The total protein was washed again with TBS for 10 minutes, incubated with secondary antibody dilution buffer (1:800) labeled with HRP at room temperature for 1 hour, decolorized with TBST at room temperature, washed three times on a shaking table for 10 minutes each time, and finally tested with ECL luminescence kit. The results were quantitatively analyzed using Gel pro4.0 gel optical density analysis software. In addition, the integrated optical density was determined.
2.7 Statistical analysis
All analyses were performed using SPSS13.0 software. The results are presented as mean ± SD. Comparison between two groups was performed using one-way ANOVA. P values of <0.05 were regarded as statistically significant.
3.1 Effect of ACh and Gal on OGD model of renal macrophages
The OGD model of renal macrophages was constructed to test the effect of miRNA-132 in renal macrophages under AKI caused by hemorrhagic shock. Macrophages subjected under hypoglycemia and hypoxia were processed for 24 hours with ACh and Gal as antagonists, and the expression levels of TNF-α, IL-1β, IL-10, TGF-β, IL-4 and IL-6 were tested in cell culture suspension. The results demonstrated that the expression levels of TNF-α, IL-1β, IL-10, TGF-β, IL-4 and IL-6 after treatment with ACh and Gal were all significantly lower than those in the group of macrophages subjected under hypoglycemia and hypoxia (P < 0.05 or P < 0.01) (Figures 1A–F).
3.2 Expression of miRNA-132 in renal epithelial cells
To further verify the effect of miRNA-132, we constructed its expression plasmid and then transfected in renal epithelial cells. We found that 3 days after its transfection, miRNA-132 was stably expressed and the fluorescence intensity was higher than that in the control group (Plasmids were not inserted into the miRNA-132 group) (Figures 2A, B)
3.3 Analysis of cell viability
Analysis of cell viability of each experimental group, using the MTT method showed that the cell viability of renal macrophages was significantly reduced in the 10%XB+OGD+miRNA-132 group compared to the 10%XB+OGD group (P < 0.01). Moreover, the cell viability of macrophages in double cell culture system with expression of miRNA-132 was similar to that of normal cells (Figure 3).
3.4 Effect of miRNA-132 on expression of TNF-α, IL-1β, IL-10, TGF-β, IL-4 and IL-6
The effect of miRNA-132 was further verified in the double cell culture system of renal macrophage–renal epithelial cell, and the expression levels of TNF-α, IL-1β, IL-10, TGF-β, IL-4 and IL-6 were analyzed in the double cell culture system, in which miRNA-132 was transfected and stably expressed. Analysis showed that the expression levels of TNF-α, IL-1β, IL-10, TGF-β, IL-4 and IL-6 in the cell suspension with transfected and stably expressed miRNA-132 were significantly lower than that in the macrophages of the hypoglycemia and hypoxia group (P < 0.05 or P < 0.01), demonstrating that miRNA-132 inhibits inflammation (Figures 4A–F).
3.5 Effect of miRNA-132 on expression levels of AChE mRNA and protein in renal macrophages
The effect of transfected miRNA-132 was investigated, and the results demonstrated that miRNA-132 expression in rat renal macrophages of double cell culture system with transfected expression plasmids was significantly higher than that in other experimental groups (Figure 5A). Moreover, the effects of transfection and expression of miRNA-132 on AChE mRNA and protein and their activities were investigated in the double cell culture system. The results demonstrated that expression of AChE mRNA was significantly inhibited by overexpression of miRNA-132 in renal macrophages (Figure 5B, ** P < 0.01). The results also showed that the expression of AChE protein in the cells was significantly inhibited (Figure 5C). Expression of AChE protein was then quantitatively analyzed (Figure 5D). The effects of transfection and expression of miRNA-132 on AChE-R mRNA in the double cell culture system. We found the expression of AChE-R mRNA was significantly inhibited by overexpression of miRNA-132 (Figure 5F, ** P < 0.01), and there was no obvious change in the expression of AChE-S. The results show that miRNA-132 regulates inflammation by inhibiting the expression of AChE-R protein in renal macrophages. This has some protective effect on renal injury (Figure 6).
It has become evident that microRNAs (miRNAs) are pivotal regulators of many biological processes, including inflammation . Our study has proven that the ACh-mediated anti-inflammatory response was increased by miRNA-132 in renal macrophage–renal epithelial cell system of AKI caused by hemorrhagic shock in rats. Alleviation of inflammatory response, neovascularization, and tissue repair, were promoted by macrophages in the specific microenvironment . The microenvironment of renal macrophages was altered resulting from the release of large number of inflammatory mediators when the kidney is chemically injured. A large nuber of inflammatory mediators, particularly some proinflammatory cytokines (e.g., TNF-α, IL-1β, IL-10, TGF-β, IL-4 and IL-6) were all activated and released, resulting in self-destructive systemic inflammation that eventually developed into MODS [13,14]. Our study found that the expression level of inflammatory factors, such as TNF-α, IL-1β, IL-10, TGF-β, IL-4 and IL-6 was significantly higher in transfected cells than those in normal cells and cells treated with antagonists, such as ACh and Gal.
The cholinergic anti-inflammatory pathway is a neural regulatory pathway, whereas synthesis and release of inflammatory factors is effectively reduced, resulting in significant inhibition of systemic and local inflammatory responses when this pathway is activated [15,16]. nAChRα7 in monocyte–macrophages is activated to subsequently inhibit the activation of monocyte–macrophage and reduce the release of pro-inflammatory cytokines, such as TNF-α. Acetylcholinesterase is not only present in neuronal cells but also in immunocytes ; the highest expression was observed in macrophages . Whenever an inflammatory response occurs, ACh is rapidly hydrolyzed by AChE secreted from macrophages to prevent the inhibitory effect of ACh on macrophage activation. MiRNA-132 expression is upregulated in the human monocyte–macrophage cell line THP-1 processed by LPS, whereas miRNA-132 acts as the ligand of immune response involved in regulation of TLR signal molecules and activation of NF-KB pathway, demonstrating the close relationship between miRNA-132 and inflammatory response . MiRNA-132 can target the 3’-UTR of AChE mRNA to inhibit AChE expression and regulate cholinergic anti-inflammatory pathway accordingly .
The hydrolytic activity of AChE is presumed to be reduced by miRNA-132 via the inhibition of AChE expression in renal macrophages, improving the microenvironment in the kidney, inducing the conversion of renal macrophages into type M2, promoting repair of renal tubule and renal interstitium and improving renal function [20,21]. Our investigation found that after the renal epithelial cells with transfection and stable expression of miRNA-132 are placed over the macrophages of a kidney injured by hypoglycemia and hypoxia, the expression levels of TNF-α, IL-1β, IL-10, TGF-β, IL-4 and IL-6 in cell culture supernatant are significantly inhibited. Moreover, the expression level of miRNA-132 in the double cell culture system is significantly higher than that in other experimental groups. The cholinergic anti-inflammatory effect of miRNA-132 results from targeted inhibition of the hydrolysis of AChE on ACh, whereas our results demonstrated that the expression level of AChE mRNA and protein in renal macrophages that stably expresses miRNA-132 is significantly reduced. Several splice variants of AChE are known (e.g., AChE-S, -R), which result in forms that differ in solubility and subcellular localization. AChE-R furthermore exhibits a unique C-terminal sequence, which is responsible for nonenzymatic actions of this variant . It is involved in proliferation, apoptosis and development of various cell types in different organs, for example brain and hematopoietic cells . In our study, we found increases in miRNA-132 can suppress AChE-R, whereas AChE-R suppression would elevate synaptic ACh, increasing the cholinergic anti-inflammatory effect.
Our study has proven that potential regulation of the inflammatory response by miRNA-132 in renal macrophages possibly plays a preventive, therapeutic, and protective role toward kidney injury caused by hemorrhagic shock.
The present study was supported by the National Natural Science Foundation of China (grant No. U1204810), the Guangdong Natural Science Foundation (grant No. 2014A030313709), Shenzhen Science and Technology Planning Project (grant No. JCYJ20130401112313541, No. JCYJ20140415162338855, No.JCYJ20150330102401099, No.JCYJ 20160425103130218, No.JCYJ20170306091335008), and Clinical and Foundation Bridge Project of Shengzhen Second Hospital (2015-16)
Cocchi M.N., Kimlin E., Walsh M., Donnino M.W., Identification and resuscitation of the trauma patient in shock, Emerg Med. Clin. North Am., 2007, 25(3), 623-642. PubMedCrossrefWeb of ScienceGoogle Scholar
Zhou B., Wang G., Peng N., He X., Guan X., Liu Y., Pre-Hospital Induced Hypothermia Improves Outcomes in a Pig Model of Traumatic Hemorrhagic Shock, Adv. Clin. Exp. Med., 2015, 24(4), 571-578. Web of ScienceCrossrefGoogle Scholar
Coca S.G., Yusuf B., Shlipak M.G., Garg A.X., Parikh C.R., Longterm risk of mortality and other adverse outcomes after acute kidney injury: a systematic review and meta-analysis, Am. J. Kidney Dis., 2009, 53(6), 961-973. CrossrefGoogle Scholar
Molitoris B.A., Levin A., Warnock D.G., Joannidis M., Mehta R.L., Kellum J.A., et al., Improving outcomes of acute kidney injury: report of an initiative Nature clinical practice, Nephrology., 2007, 3(8), 439-442. PubMedGoogle Scholar
Van Ginderachter J.A., Movahedi K., Hassanzadeh Ghassabeh G., Meerschaut S., Beschin A., Raes G., et al., Classical and alternative activation of mononuclear phagocytes: picking the best of both worlds for tumor promotion, Immunobiology., 2006, 211(6-80), 487-501. PubMedCrossrefGoogle Scholar
Wang H., Yu M., Ochani M., Amella C.A., Tanovic M., Susarla S., et al., Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation, Nature, 2003,421(6921), 384-388. CrossrefPubMedGoogle Scholar
Yoshikawa H., Kurokawa M., Ozaki N., Nara K., Atou K., Takada E., et al., Nicotine inhibits the production of proinflammatory mediators in human monocytes by suppression of I-kappaB phosphorylation and nuclear factor-kappaB transcriptional activity through nicotinic acetylcholine receptor alpha7, Clin. Exp. Immunol., 2006, 146(1), 116-123. PubMedCrossrefGoogle Scholar
Mann M., Mehta A., Zhao J.L., Lee K., Marinov G.K., Garcia-Flores Y., et al., An NF-kappaB-microRNA regulatory network tunes macrophage inflammatory responses, Nat. Commun., 2017, 8, 851. CrossrefPubMedGoogle Scholar
Wang Y., Wang Y., Cao Q., Zheng G., Lee V.W., Zheng D., et al., By homing to the kidney, activated macrophages potently exacerbate renal injury, Am. J. Pathol., 2008, 172(6), 1491-1499. PubMedCrossrefWeb of ScienceGoogle Scholar
van Maanen M.A., Vervoordeldonk M.J., Tak P.P., The cholinergic anti-inflammatory pathway: towards innovative treatment of rheumatoid arthritis, Nat. Rev. Rheumatol., 2009, 5(4), 229-232. Web of ScienceCrossrefPubMedGoogle Scholar
Hilderman M., Qureshi A.R., Al-Abed Y., Abtahi F., Lindecrantz K., Anderstam B., et al., Cholinergic antiinflammatory pathway activity in dialysis patients: a role for neuroimmunomodulation? Clin. Kidney. J., 2015, 8(5), 599-605. CrossrefGoogle Scholar
Han Z., Li L., Wang L., Degos V., Maze M., Su H., et al., Alpha-7 nicotinic acetylcholine receptor agonist treatment reduces neuroinflammation, oxidative stress, and brain injury in mice with ischemic stroke and bone fracture, J. Neurochem., 2014, 131(4), 498-508. CrossrefPubMedWeb of ScienceGoogle Scholar
Klegeris A., Budd T.C., Greenfield S.A., Acetylcholinesterase-induced respiratory burst in macrophages: evidence for the involvement of the macrophage mannose-fucose receptor, Biochim. Biophys. Acta, 1996., 1289(1), 159-167. CrossrefPubMedGoogle Scholar
Taganov K.D., Boldin M.P., Chang K.J., Baltimore D., NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses, Proc. Natl. Acad. Sci. U S A., 2006, 103(3), 12481-12486. PubMedCrossrefGoogle Scholar
Shaked I., Meerson A., Wolf Y., Avni R., Greenberg D., Gilboa-Geffen A., et al., MicroRNA-132 potentiates cholinergic antiinflammatory signaling by targeting acetylcholinesterase, Immunity., 2009, 31(6), 965-973. CrossrefGoogle Scholar
Mishra N., Friedson L., Hanin G., Bekenstein U., Volovich M., Bennett E.R., et al., Antisense miR-132 blockade via the AChE-R splice variant mitigates cortical inflammation, Sci. Rep., 2017, 7, 42755. CrossrefWeb of SciencePubMedGoogle Scholar
Blohberger J., Kunz L., Einwang D., Berg U., Berg D., Ojeda S.R., et al., Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions? Cell Death Dis., 2015, 6(10), e1685. . CrossrefWeb of SciencePubMedGoogle Scholar
About the article
Published Online: 2018-03-20
Conflict of interest: Authors state no conflict of interest.
Citation Information: Open Chemistry, Volume 16, Issue 1, Pages 176–183, ISSN (Online) 2391-5420, DOI: https://doi.org/10.1515/chem-2018-0019.
© 2018 Ming Wu et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License. BY-NC-ND 4.0