key: cord-1030955-x5e9nfde
authors: Li, Xiangyun; Yu, Jianbo; Gong, Lirong; Zhang, Yuan; Dong, Shuan; Shi, Jia; Li, Cui; Li, Yuting; Zhang, Yanfang; Li, Haibo
title: Heme oxygenase-1(HO-1) regulates Golgi stress and attenuates endotoxin-induced acute lung injury through hypoxia inducible factor-1α (HIF-1α)/HO-1 signaling pathway
date: 2021-01-23
journal: Free Radic Biol Med
DOI: 10.1016/j.freeradbiomed.2021.01.028
sha: be89ea8c8f58a0856e24c424cc211e8cce2ca89f
doc_id: 1030955
cord_uid: x5e9nfde

Sepsis caused acute lung injury (ALI) is a kind of serious disease in critically ill patients with very high morbidity and mortality. Recently, it has been demonstrated that Golgi is involved in the process of oxidative stress. However, whether Golgi stress is associated with oxidative stress in septic induced acute lung injury has not been elucidated. In this research, we found that lipopolysaccharide (LPS) induced oxidative stress, apoptosis, inflammation and Golgi morphology changes in acute lung injury both in vivo and in vitro. The knockout of heme oxygenase-1(HO-1) aggravated oxidative stress, inflammation, apoptosis and reduced the expression of Golgi matrix protein 130 (GM130), mannosidase Ⅱ, Golgi-associated protein golgin A1 (Golgin 97), and increased the expression of Golgi phosphoprotein 3 (GOLPH3), which caused the fragmentation of Golgi. Furtherly, the activation of hypoxia inducible factor-1α (HIF-1α)/HO-1 pathway, attenuates Golgi stress and oxidative stress by increasing the levels of GM130, mannosidase Ⅱ, Golgin 97, and decreasing the expression of GOLPH3 both in vivo and in vitro. Therefore, the activation of HO-1 plays a crucial role in alleviating sepsis-induced acute lung injury by regulating Golgi stress, oxidative stress, which may provide a therapeutic target for the treatment of acute lung injury.

The Golgi apparatus acts as an important part of the endomembrane system and exists in all eukaryotic cells. In most higher eukaryotic cells, the Golgi consists of a series of flattened cisternae organized into ribbon-like stacks that are closely arranged to each other [1] .These interconnected highly organized stacks of cisternae play an crucial role in the secretory and the endocytic pathways [2] .The maintenance of Golgi organization depend on the large coiled-coil proteins which are known as 'golgins' [3] . These glogins include a variety of members, such as GM130, Golgin-160, Giantin and others, which are necessary for vesicular transport, and the maintenance of Golgi structure. It has been demonstrated that Golgi complex is relevant to the ion homeostasis, cell apoptosis, antioxidation, and stress sensing [4] . The integrated structure and steady function of Golgi apparatus could be impaired under oxidative stress, DNA damage and pro-apoptotic conditions, which may even induce Golgi fragmentation and cell apoptosis [5, 6] . Therefore, the Golgi complex has become a new candidate for the research and therapy about oxidative stress-associated diseases.

Sepsis-induced acute lung injury(ALI) is recognized as life-threatening diseases that can aggravate to more severe acute respiratory distress syndrome(ARDS) [7, 8] . Since the pandemic of coronavirus disease 2019 , about 5% patients with COVID-19 progress to ARDS, J o u r n a l P r e -p r o o f septic shock and /or multiple organ failure [9, 10] .Therefore, more and more research are focusing on the treatment of acute lung injury or ARDS. Nowadays, it has been demonstrated that septic lung injury associated with a lot of organelles dysfunction. For example, sepsis induced acute lung injury and ARDS is related to the disruption of mitochondrial dynamics and enhanced endoplasmic reticulum stress [11, 12] . However, whether acute lung injury can induce the dysfunction and fragmentation of Golgi has not been certified. Therefore, we hypothesized that LPS could induce Golgi stress by disturbing the morphology homeostasis in vivo and in vitro.

Heme oxygenase-1, a stress inducible protein, play an critical effect on signaling and cytoprotective activities in different organs and tissues, as well as its degradation product carbon monoxide(CO), biliverdin and iron [13] . Recently, many studies have shown that HO-1 is involved in maintaining the homeostasis of organelles in cells, such as mitochondria, endoplasmic reticulum and so on [14, 15] . Our previous research has demonstrated that HO-1 protect cells from inflammation and oxidative stress by improving mitochondrial dynamics in endotoxin induced acute lung injury in vivo and in vitro [12, [16] [17] [18] . However, the function of HO-1 on Golgi stress has not been studied. Therefore, in this study, we aimed to identify the relationship between HO-1 and Golgi stress and their mechanisms in LPS-induced ALI. In order to find out the function of HIF-1α α α α/HO-1 signaling pathway in Golgi stress in before sacrificing. All the procedures were performed according to National Institutes of Health guidelines. The mice were fed with standard laboratory diet and water at 25℃, and lived in the animal room with 12h light-dark cycle (Fig. 1C) . 

The left lung tissues were fixed in 10% formaldehyde, embedded in paraffin, sectioned at 5μm thick and stained with hematoxylin and eosin(H&E). H&E-stained slides were observed with a light microscope (Olympus, Nagano, Japan) after dehydration and sealing. MLE12 cells were pretreated with 100μM DMOG (dissolved in DMSO) for 24h, then stimulated with 5μg/ml LPS for another 24h before harvesting (Fig. 1B ).

Total RNA in tissue and cells was isolated by using Trizol (Invitrogen Life Technologies, Carlsbad, CA). Then cDNA was created using reverse transcriptase (Thermos Fisher, EP0442).

AceQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd) was used for real time PCR.

Gene-specific primers used in this experiment were shown in J o u r n a l P r e -p r o o f

The lung tissue slices and the cells were fixed with 4% paraformaldehyde for 10 min, and then washed with PBS for three times. After incubating the primary antibody overnight, the tissue and slides were incubated with fluorescein-conjugated secondary antibody for 1h. Anti-GM130 antibody (610822; BD Bioscience) and DAPI were used for Golgi and nuclear structure, respectively. Fluorescence pictures were obtained with Nikon confocal fluorescence microscope. 

The proteins were extracted from right lung tissue using a total and nuclear protein isolation kit (Thermo Fischer Scientific, Inc.). After the cells were treated with LPS(5ug/ml) for 24 hours, cells were washed with phosphate-buffered saline and then homogenized in RIPA buffer (Beyotime, P0013B). The concentrations of these proteins were detected using a BCA protein assay kit (Thermo, USA). Equal amounts of protein were subjected to 10% SDS-PAGE gel and then were transferred to a PVDF membrane (Millipore Cat #IPVH00010). Membranes were blocked with 5% 

The values are expressed as mean ±standard deviation (SD). T-test and nonparametric tests were used for comparisons between two groups. One-way ANOVA was used to make comparisons among multiple groups, followed by the Bonferroni correction post hoc test. Graph Prism 7.0 software (GraphPad Software, USA) was used for statistical analysis. Statistical significance was considered at P<0.05.

C57BL/6J mice were exposed to LPS (10mg/kg) or PBS (control) for 12 hours and the BALF was obtained for cell differential and ELISA. HE staining was used to evaluate the pathological changes in our study. These slices showed that LPS induced the thickening of alveolar septum, 2F ). TUNEL positive cell numbers were significantly higher in LPS group than that in control group (Fig. 2F ). Therefore, we concluded that LPS induced oxidative stress, inflammation, apoptosis along with acute lung injury in WT mice.

GM130, localized on the cis side of the Golgi cisternae, is involved in the disassembly and reassembly of the Golgi during the process of mitosis, thus it regarded as a specific marker for the Golgi apparatus [28] . Golgin97,which is located in the trans-Golgi network, plays crucial roles in vesicle trafficking and maintaining the Golgi integrity [29] . Mannosidase Ⅱ is related to oligosaccharide processing in N-linked glycoprotein biosynthesis and regarded as a marker for medial-Golgi compartments [30] . It has been found that GOLPH3 is one of Golgi stress inducible proteins in oxygen-glucose deprivation and reoxygenation treated N2A cells [31] . Further studies on the morphology changes of Golgi was performed using the double immunofluorescence(IF)

with FITC-labeled GM130-antibody(red) and DAPI(blue, nuclear staining).As shown in 

HO-1 is one of inducible proteins in many diseases under the insults of oxidative stress and inflammatory response [32] . It has been demonstrated that the protective effect of HO-1 is related to maintaining the homeostasis of organelles in cells, such as mitochondria, endoplasmic reticulum and so on [14, 15] . In this research, we try to find out the effect of HO-1 on Golgi stress HE staining from Fig.3B showed that the knockout of HO-1 exaggerated lung injury. Albumin and IL-6 in BALF were higher in HO-1 knockout mice, when compared with LPS(Cre) group ( Fig.3C-D) . ROS levels in lung tissue also increased after the knockout of HO-1 (Fig. 3E) . Total leukocytes number in BALF increased significantly in HO-1KO group after the treatment of LPS (Fig. 3F ). Cell differential results from BALF showed that the knockout of HO-1 increased the number of macrophages and neutrophils number in BALF (supplemental data Fig. 1D -F). More apoptosis cells were found after the knockout of HO-1 in lung tissue (Fig.3G ). Further studies on Golgi structure were performed by immunofluorescence (Fig. 3H ). There were more Golgi fragmented into a lot of punctate structures in the cytoplasm in HO-1KO group (Fig. 3H-I) . in lung tissue of mice (Fig.6B-H) . Therefore, the activation of HIF1α/HO-1 signaling pathway attenuated acute lung injury and reduced Golgi stress by increasing the expression of Golgi structure proteins and decreased Golgi stress protein in vivo.

In this research, we treated MLE12 cells with 100μM DMOG for 24h before the treatment of 5μg/ml LPS for another 24h to activate the expression of HIF-1α /HO-1 signaling pathway. The results showed that LPS inhibited cell viability and SOD activity and enhanced the expression of ROS levels( Fig. 7A-C) . To the contrary, the treatment of DMOG improved cell viability, as well as SOD activity, and decreased the expression of ROS in vitro ( Fig. 7A-C) . 

Golgi apparatus dysfunction has been increasingly regarded as one crucial factor to the pathogenesis of human disease [31, 43] .Our research provide strong evidence that LPS induced The Golgi apparatus is one of the most complicated organelles in the cells. More and more researches are focused on the morphology and function changes of Golgi in some oxidative stress related diseases [1, 5, 6] . However, the molecular mechanisms and its relative physiological functions of the Golgi stress has not been clarified. The interplay among different organelles, such as ER, Golgi, mitochondria, is intricate and finding their communication among them would also be of great importance. Therefore, it would be of great interest to find out more elaborate J o u r n a l P r e -p r o o f regulations in this aspect, which will provide some new understanding and therapy methods for more diseases in future.

Golgi stress is involved in the progression of endotoxin-induced acute lung injury both in vivo and in vitro. HO-1 protects against septic lung injury by inhibiting Golgi stress, increasing the expression of Golgi structure related proteins, and reducing oxidative stress and inflammation. HO-1siRNA for 24h and then incubated with 5μg/ml LPS for another 24h before harvesting them.

To explore the function of HIF-1α/HO-1 pathway in Golgi stress in vitro, MLE12 cells firstly were pre-treated with 100μM DMOG (dissolved in DMSO) for 24h before 5μg/ml LPS incubation for another 24h. Fig.1C . In order to find out the role of HIF-1α/HO-1 pathway in Golgi stress in vivo, C57BL/6 mice were pretreated with DMOG 25mg/ml, 300μl by intraperitoneal injection for 24 hours, and then stimulated with LPS for 12 hours before sacrificing. HE staining for lung tissue in WT mice after the treatment of 10mg/kg LPS and PBS for 12h ( Fig.   2A ). Albumin and IL-6 were detected in BALF by ELISA (Fig. 2B-C) . ROS levels in lung tissue ( Fig. 2D) . Total leukocytes number in BALF (Fig. 2E) . TUNEL staining for apoptosis in lung tissue (Fig. 2F ). Golgi structure changes in confocal ( Fig. 2G-H) . mRNA levels of Golgi morphology related proteins and GOLPH3 ( Fig. 2I-L) . Golgi structure changes in electron microscope (Fig. 2M) . * P<0.05 vs. control, N=3 at least for each group. Data were presented as mean ±SD, and were analyzed by t-test. 3C-E). Total leukocytes number in BALF (Fig. 3F) . TUNEL staining for apoptosis in lung tissue (Fig.3G) . Immunofluorescence staining to see Golgi morphology changes in lung tissue ( Fig.   3H -I). qPCR results showed the mRNA expression levels of Golgi morphology related proteins and GOLPH3 in vivo (Fig. 3J-M) . Golgi structure changes in electron microscope (Fig. 3N) . * P<0.05 vs. control, N=3 at least for each group. Data were presented as mean ±SD, and were analyzed by t-test. Cell viability significantly decreased when the cells were incubated with LPS for 24h at the concentration of 1μg/ml,5μg/ml and 10μg/ml (Fig. 4A) . HO-1 siRNA transfection(50pM for 24h) were used to knockdown the expression of HO-1 and then the cells were incubated with 5μg/ml LPS for another 24h.The cell viability was reduced after the treatment of LPS when compared with the control group. Furtherly HO-1 knockdown group showed much lower cell viability (Fig.   4B ). ROS and SOD activity in MLE12 cells (Fig. 4C-D) . Fluorescent staining results show Golgi morphology changes and Golgi fragmentation numbers for each field in vitro (Fig. 4E) . Golgi structure changes were evaluated by electron microscope (Fig.4F) J o u r n a l P r e -p r o o f The C57BL/6 mice were pretreated with DMOG to activate HIF-1α/HO-1 signaling pathway and then treated with LPS to induce acute lung injury. The lung tissue injury levels by HE staining (Fig.6A) . The western blot to show the protein levels of HIF-1α, HO-1, GM130, mannosidase Ⅱ, Golgin97 and GOLPH3 in lung tissue (Fig. 6 B-H) . * P<0.05 vs. control, # P<0.05

vs. LPS group. N=3 at least for each group. Data were presented as mean ±SD, and were analyzed by one-way analysis of variance (ANOVA), followed by the Bonferroni correction post hoc test.* 

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