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The miR-15a affected the development of sepsis and septic shock by suppression BCL-2


 Background: This study aimed to investigate the mechanism of microRNA-15a (miR-15a) in the development of sepsis and septic shock. Methods: Sepsis and septic shock rat models were constructed by intraperitoneal injection of E.coli endotoxin (LPS). The real-time polymerase chain reaction (RT-PCR), Enzyme-Linked ImmunoSorbent Assay (ELISA), ematoxylin-eosin (HE) and Masson staining, TdT-mediated dUTP Nick-End Labeling (TUNEL), as well as Western blot analysis were performed to reveal the expression of microRNA-15a and changes in sepsis/septic shock myocardial cells or tissue. The rat sepsis model (sepsis group and septic shock group) was successfully established. Results: The results of HE and Mason staining showed that myocardial tissue damage gradually deepened with the progression of sepsis. Moreover, the serum levels of creatinine kinase-mb (CK-MB) and cardiac troponin I (cTnI) in model groups were significantly increased than those in control group. In addition, the RT-PCR analysis showed that miR-15a was up-regulated in model groups. Furthermore, luciferase reporter gene assay showed that 3'-UTR was the binding site of BCL-2 to miR-15a. Finally, the TUNEL and Western blot showed the cardiomyocyte apoptosis in model group. Conclusions: The overexpression of miR-15a might take part in the progression of LPS-induced sepsis and septic shock via suppressing Bcl-2 expression. Furthermore, myocardial markers such as CK-MB and cTnI might be biomarkers for sepsis progression.


Background
Sepsis is life threatening organ dysfunction caused by a dysregulated host response to infection 1 . As the subset of sepsis, septic shock is defined in which underlying circulatory and cellular or metabolic abnormalities, and are profound enough to increase mortality substantially 2 . Generally, 40-60% sepsis patients have multiple organ dysfunction 3 .
Among these, myocardial dysfunction is one of the main predictors of poor outcome in septic patients, with mortality rates next to 70% 4 . Although novel therapeutic pathways targeting organ dysfunction hold renewed promise for septic, the latest estimates of incidence in the United States, Europe, and the United Kingdom is as high as 1/1000 of the population 5 . Therefore, studying the pathogenesis of sepsis and its subset septic shock is vital for the clinical treatment.
Myocardial inhibition is a recognized manifestation of organ dysfunction in sepsis, and myocardial apoptosis is a key component of its development 6 . During this process, RNA (microRNAs) are commonly used as fingerprints for sepsis and proved to be potential septic biomarkers 7 . The overexpression of miRNAs increase lipopolysaccharide (LPS)induced rat survival and inhibit LPS-induced cardiomyocyte apoptosis 8 , which can be used as an independent therapeutic target for sepsis 9 . Aa a member of miRNAs, miR-15a is confirmed to participate in a variety of biological processes including proliferation and apoptosis 10,11 . Wang et al. indicated that the up-regulation of miR-15a inhibited LPSinduced biological pathways in sepsis 12 . Not only miR-15a can be used as a biomarker to distinguish sepsis from systemic inflammatory response syndrome (SIRS) 13 , but bacterial infection and/or bacterial LPS increase the level of miR-15a/16 in macrophages 14 . In clinical treatment, myocardial depression is always combined with the variation of indicators such as cardiac troponin I (cTnI), procalcitonin (PCT) and creatinine kinase-mb (CK-MB) in patients until death or recovery 15,16 . Importantly, a previous study shows that myocardial apoptosis-regulating gene B cell lymphoma 2 (Bcl-2) might be used as an indicator during the process of myocardial injury in sepsis 17 . Members of Bcl-2 gene family have a central role in regulating programmed cell death by controlling proapoptotic and anti-apoptotic intracellular signals 18 . In previous study, the mice model has been successfully used to examine the role of Bcl-2 family members on the regulation of sepsis induced splenic dendritic cells and macrophages survival 19 . Thus, selective inhibition of specific anti-apoptotic Bcl-2 family proteins represents an exciting therapeutic opportunity for sepsis and septic shock. However, the biological function of Bcl-2 and miR-15a in sepsis and septic shock had not yet been thoroughly studied.
In this study, the LPS-induced sepsis and septic shock rat model were established. The real-time polymerase chain reaction (RT-PCR), Enzyme-Linked ImmunoSorbent Assay (ELISA), ematoxylin-eosin (HE) and Masson staining, TdT-mediated dUTP Nick-End Labeling (TUNEL), as well as Western blot analysis were performed to reveal the expression of microRNA-15a and changes in myocardial cells or tissue. Our findings may reveal the detail roles of miR-15a on neural damages of sepsis and septic shock, and provide new insights into the underlying mechanisms responsible for the treatment of sepsis and septic shock.

Rat model construction and grouping
A total of 60 male Sprague Dawley (SD) rats (180-200 g, 6 weeks, SPF grade) were purchased from Experimental Animal Center of the Chinese Academy of Sciences. After one week standard feeding, all rats were randomly divided into control group (n = 12, intraperitoneal injection of 10 mg/kg PBS), sepsis group (n = 24, intraperitoneal injection of 10 mg/kg LPS (O55:B5; Sigma, St Louis, MO, USA)) and septic shock group (n = 24, intraperitoneal injection of 10 mg/kg LPS (O55:B5; Sigma, St Louis, MO, USA)). Systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) (monitored by a pressure transducer) were used to calculate mean arterial pressure (MAP) with MAP = DBP + (1/3 (SBP -DBP)). Compared with the baseline value, the MAP value decreased by less than 30%, equal to 30% and more than 30% was considered as sepsis model, shock model and septic shock model respectively. This study was approved by the ethics committee of

Specimen collection
Blood (4ml/each sample) was collected from the common carotid artery intubation. One sample of blood was treated with anticoagulant (heparin) and centrifuged to obtain plasma. The other sample of natural liquid was precipitated by centrifugation to obtain serum. The plasma and serum were separately obtained for the detecting biochemical indicators. After the rats were killed, the heart tissues were removed and fixed at 10% formaldehyde with the size of 1.0 mm 3 . The rest of the heart tissues were stored at -80 ℃.

CK-MB, cTnI and PCT detection
The CK-MB and PCT of the three groups were measured by ELISA based on the kit instructions (Wuhan Yousheng Technology Co., Ltd., R&D Company of the United States).
Meanwhile, the cTnI levels of three groups were measured using a Beckman-Coulter ACCESS 2 automatic microparticle chemiluminescence immunoassay analyzer. All specific operations were strictly in accordance with the instructions.

RT-PCR assay
Total RNAs were extracted from cells in different groups using TRizol reagent (Invitrogen, USA), and reverse transcripted using MBI (San Francisco, CA, USA). Ultraviolet spectrophotometer was used to determine the concentration and purity of the RNA. The qRT-PCR was performed on ABI7500 (Thermo Fisher Scientific), and the amplification primer sequences of each gene and their primers were shown in Table 1

Western blot assay
The expression of proteins in myocardial cell were detected by Western blot. Total proteins of myocardial cell from different groups were extracted by RIPA lysis buffer (Thermo Scientific, USA), and determined by Bradford method. After centrifugation, proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred to a polyvinylidenefluoride membrane (Roche). Then the membrane was blocked with 50g/L skim milk for 2 h, and incubated with goat anti-rat primary antibody (1:5000, Abcam, USA) overnight at 4°C. Then, the membrane was incubated with HRP-labeled secondary antibody IgG (1:5000, Jinqiao Biotechnology, China) for 2 h at 37°C. Finally, the protein was colored by DAB and imaged by image analysis system for analysis.
After the study, all rats were euthanized. The right hand held the rat tail and pull it back, and the left thumb and forefinger pressed down firmly on the rat head at the same time.
The external force was used to dislocate the cervical spine of the rat, and the spine and the brain were disconnected. This method can quickly lose consciousness and reduce pain of experimental animals, which is a commonly used method for euthanasia of small experimental animals.

HE staining
After the rats were executed, the right hind limb was fixed with 10% neutral formaldehyde solution for 24h. Then the decalcification process was performed for 30 days by using 10% Ethylenediaminetetraacetic acid (EDTA), followed by routine dehydration, transparency, paraffin immersion, embedding and coronal section. The sections were dewaxed and dehydrated with ethanol of different gradient concentration, then stained with Hematoxylin-eosin (HE). The pathological changes were observed under light microscope.

Masson staining
The paraffin sections need to undergo xylene dewaxing and alcohol gradient dehydration before they were subjected to Masson staining. Then, the sections were dyed with saturated alcohol bitter acid for 1h, and hematoxylin stained for 5-10min. After differentiation by hydrochloric acid and alcohol, the sections were stained with Ponceau acid fuchsin solution for 10 minutes and 1% phosphomolybdic acid for 5 minutes in turn, and then they were immersed in bright green solution. After gradient alcohol dehydration and xylene infiltration, the paraffin sections were sealed by neutral resin. The change of collagen content was observed under light microscope, the collagen and non-collagen components were distinguished by gradation adjustment. Moreover, the HP IAS2000 computer image analysis system was used to measure myocardial collagen volume fraction (CVF) and myocardial perivascular collagen area (PCVA).
Detection of cell apoptosis TUNEL assay was performed to detect the myocardial cell apoptosis in rats from each group. T The rat heart was fixed with 10% formaldehyde, embedded in paraffin, then dewaxed and hydrated with a gradient of ethanol. DNA 3-OH terminal was labeled by terminal deoxynucleotidyl TUNEL, and apoptotic cells were detected by in situ fluorescence labeling. According to the TUNEL kit (ROCHE, Swiss) instructions, the apoptotic cardiomyocytes was with red fluorescence, while all nucleated cells was with blue fluorescence after DAPI staining. Finally, four fields of ischemic area were randomly selected, and the total number of cells and the number of apoptotic cells in each field were counted, and the apoptotic index (AI) was calculated.

Luciferase reporter gene assay
The target sites for Bcl-2 and miR-15a were determined by Target Scan, and the mutant and wild sequences were designed according to the predicted results. The Bcl-2 mutant sequence and the wild sequence fragment were cloned and bound to the PGL-3 vector.
The mutated sequence was co-transfected into 293T cells with miR-15a mimics or the miR-15a negative control, and named as MT + mimics group and MT + NC group, respectively. In addition, 293T cells were transfected with wild-type sequences combined with miR-15a or its' negative control. After transfection for 48 hours, fluorescent activity of the cells was detected by luciferase kit (Yuanping Hao Biotechnology Co., Ltd., China).

Statistical methods
Data analysis was performed using GraphPad Prism v5.01 statistical software, and all data were expressed as mean ± standard deviation (SD). The comparison between groups was analyzed by t-test. P < 0.05 was considered statistically significant.

Hemodynamics in rats
The sepsis model and the shock model were successfully established at 12 and 24 hours LPS treatment, followed by the hemodynamics analysis. The result showed that the SBP, DBP, and MAP of rats in control group was 127 ± 4 mmHg, 75 ± 3 mmHg, and 93 ± 3 mmHg, respectively. After LPS injection for 20 hours, the blood pressure was dropped by about 30%, and the rat entered the shock state. The SBP ( Figure 1A), DBP ( Figure 1B) and MAP ( Figure 1C) levels after LPS injection were significantly lower than those of control group (P < 0.05). Meanwhile, HR level ( Figure 1D) was significantly higher than that of control group (P < 0.05).

Pathological changes of cardiac tissue
The results of HE staining showed that the myocardial structure of the control group was intact and clear. Meanwhile, the myocardial fibers were closely arranged and no obvious necrosis was observed in control group. The normal structure of the heart was destroyed, and the myocardial fibers were sparse and loosely arranged in sepsis group and septic shock group. Furthermore, the nucleus of myocardial cell was condensed and ruptured.
Compared with the septic shock group, the degree of lesions was significantly reduced in sepsis group (Figure 2A).
Masson staining showed that the cardiomyocytes were arranged tightly and neatly, and the myocardial fibers were arranged tightly in the control group ( Figure 2B). The degree of lesion in the sepsis group was significantly lower than that in the septic shock group (P < 0.05). Compared with the control group,, the CVF and PCVA level in the sepsis group and the septic shock group were increased significantly (all P < 0.05). Furthermore, the level of CVF ( Figure 2C) and PCVA ( Figure 2D) in septic shock group was significantly higher than the sepsis group (all P < 0.05).

The expression of CK-MB, cTnI and PCT
Compared with control group, serum CK-MB ( Figure 3A) and cTnI ( Figure 3B) in sepsis group and septic shock group were significantly higher than those in control group (all P < 0.05), and septic shock group was significantly higher than that in sepsis group (P < 0.05). Furthermore, ELISA was used to detect changes in serum PCT content ( Figure 3C).
The results showed that the serum PCT level of the control group was very low, and the serum PCT level of the sepsis group was significantly higher than that of the control group (P < 0.05). Meanwhile, the serum PCT level in septic shock group was significantly higher than that in sepsis group (P < 0.05). These results suggested that microRNA-15a might involve in the pathogenic process from sepsis to septic shock, and the overexpression of microRNA-15a might decrease with the development of sepsis in rats.

Expression of miR-15a and the association with Bcl-2
Real-time PCR was used to detect the expression of miR-15a in control group, sepsis group and septic shock group ( Figure 4A). The result showed that compared with control group, the expression of miR-15a were significantly increased in both sepsis and septic shock group (all P < 0.05). Meanwhile, compared with sepsis group, the expression of miR-15a was significantly lower in septic shock group (P < 0.05).
The Target Scan results showed that the binding site of Bcl-2 and miR-15a was the 3'-UTR region. Additionally, there was no obvious difference in luciferase activity between MT + mimics group and MT + NC group, but the activity of luciferase in WT + mimics group was significantly lower than that in WT + NC group (P < 0.01, Figure 4B). These results indicated that miR-15a might affect myocardial apoptosis in sepsis and septic shock animals by targeting BCL-2.

Changes of myocardial apoptosis index
The results of TUNEL assay showed that the apoptosis index of model group (sepsis group and septic shock group) was significantly higher than those of the control group (all P < 0.05), while the apoptosis index of septic shock was significantly higher than that of the sepsis group (P < 0.05) ( Figure 5A). Western blot results showed that the expression of glucose regulatory protein 78 (GRP78) in cardiac tissue of model group (sepsis group and septic shock group) was significantly higher than that of control group, while the expression of GRP78 in septic shock group was significantly higher than that in sepsis group (all P < 0.05) ( Figure 5B). Furthermore, after LPS injection, the expression of BCL-2 decreased significantly either between model group (sepsis group and septic shock group) and control group (all P < 0.05), or between sepsis group and septic shock group (P < 0.05) ( Figure 5C).

Discussion
Sepsis remains a common, expensive, and deadly problem throughout the world 21 . To explore the influencing factors such as miR-15a in the progression of sepsis, the rat sepsis model (sepsis group and septic shock group) was established in current study. The results of HE and Mason staining showed that myocardial tissue damage gradually deepened with the progression of sepsis. Moreover, the serum levels of CK-MB, cTnl and PCT in model groups were significantly increased than those in control group. In addition, the RT-PCR analysis showed that miR-15a was up-regulated in model groups. Furthermore, luciferase reporter gene assay showed that 3'-UTR was the binding site of BCL-2 to miR-15a. Finally, the TUNEL and Western blot showed the cardiomyocyte apoptosis in model group.
MiR-15ais a tumor suppressor that inhibiting cell proliferation and promote cell apoptosis in cancer progression. Serum miR-15a level can be used as a biomarker that distinguish sepsis from systemic inflammatory response syndrome in human subjects 22 . A previous study shows that miR-15a is up-regulated in the serum of neonatal sepsis patients and inhibits the LPS-induced inflammatory pathway 23 . Actually, the biological function of miR-15a is realized by targeting a variety of oncogenes including Bcl-2 24 . In chronic lymphocytic leukemia, miR-15a has a tumor suppressor function by targeting the Bcl-2 oncogene 25 . In fact, overexpression of Bcl-2 has a protective effect on apoptosis in septic cells 26 . A previous study indicates that the ability of Bcl-2 overexpression to improve survival in patients with sepsis is partly related to the prevention of changes in spleen transcriptional responses caused by sepsis 27 . Hu et al. showed that that the decreasing expression of Bcl-2 was beneficial to reduce cardiomyocyte apoptosis 28 , which further indicating that miR-15aaffected cardiomyocyte apoptosis by regulating Bcl-2. In this study, the RT-PCR analysis showed that miR-15a was up-regulated in both sepsis group and septic shock group. Meanwhile, the luciferase reporter gene assay showed that the down-regulation of Bcl-2 was a target gene of the up-regulated miR-15a. Thus, we speculated that the overexpression of miR-15a might take part in the progression of sepsis and septic shock via suppressing Bcl-2 expression.
Apoptosis was a process of inductive destruction of non-necrotizing cells and suicide 29 . It play an important role in the pathogenesis of sepsis 30 . During this process, many proteins had been studied to have an apoptotic effect. GRP78 is a heat shock protein present in all cells and a widely used marker of endoplasmic reticulum stress 31 . Wei et al. suggested that blocking inflammatory responses and inhibiting cardiomyocyte apoptosis by certain proteins including CTRP3 could affect the process of myocardial protein in septic rats 32 .
Under hypoxic conditions, the presence of peptide-bound GRP78 can inhibit cardiomyocyte apoptosis 33 . In this study, the cardiomyocyte apoptosis index GRP78 was significantly upregulated in sepsis and septic shock group, indicating that there might be apoptosis of cardiomyocytes in patients with sepsis and septic shock. Furthermore, a previous study indicates that the cute physiological can be performed on cTnI and CK-MB to evaluate the predictive value of cardiac markers in sepsis patients 34 . A Jain et al. revealed that cTnI was independent of the degree of septic myocardial dysfunction in patients with septic shock 35 . Combined detection of cTnI and CK-MB is proved to be more accurately in evaluating the progression of sepsis 36 . Wu et al. found that plasma levels of cTn1, CK-MB and PCT in the myocardial injury group were remarkably higher in children with sepsis myocardial injury than that in the non-myocardial injury group 37 . Since the CK-MB and cTnI levels were increased with the development of sepsis 38,39 , reducing serum CK-MB and cTnI levels was beneficial to improve myocardial damage in sepsis 40 . In the current study, the ELISA assay showed that the CK-MB and cTnI levels were highly expressed in sepsis and septic shock group. Thus, we speculated that myocardial markers including CK-MB and cTnI might be used as biomarkers for sepsis progression.

Conclusions
In conclusion, the overexpression of miR-15a might take part in the progression of LPSinduced sepsis and septic shock via suppressing Bcl-2 expression. Furthermore, myocardial markers such as CK-MB and cTnI might be biomarkers for sepsis progression.

Abbreviations
The real-time polymerase chain reaction RT-PCR Enzyme-Linked ImmunoSorbent Assay ELISA ematoxylin-eosin HE   Figure 1 Changes of blood pressure and heart rate in rats after lipopolysaccharide (LPS) injection. A, changes in systolic blood pressure; B, changes in diastolic blood pressure; C, changes in mean arterial pressure (MAP); D, changes in heart rate. *, P < 0.05, compared with control group.

Figure 2
The results of myocardial pathological changes and myocardial collagen analysis.

Supplementary Files
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