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Introduction

Paraquat is a type of bipyridine compound that is widely used as a herbicide (1). Self-poisoning with pesticides is a major public health problem in developing countries (2). As a result of its widespread use, paraquat is regarded as the main herbicide involved in accidental and intentional poisoning (3). Paraquat poisoning can be fatal after ingestion, and its mortality rate is 80% after confirmed exposure (4,5). Multiple organ dysfunction syndrome (MODS) is the main cause of mortality induced by paraquat poisoning (6).

MODS occurs in patients with severe infection or trauma, triggering the release of inflammatory mediators (7). The dysregulation of the inflammatory response plays a crucial role in the development of MODS (8,9). Anti-inflammatory treatment is the primary therapy for the early treatment of paraquat poisoning (5). A number of genes that are differentially expressed during the occurrence and development of inflammation are candidate targets for gene therapy, including some microRNAs (miRNAs/miRs) (10). A recent study reported that miR-146a can regulate the occurrence and immune response of lung injury induced by paraquat poisoning, and that interleukin (IL)-6 is involved in the regulation of this process (11).

IL-10 is a pluripotent anti-inflammatory cytokine that plays a crucial role in the immune response (12). He et al (13) reported that IL-10 mRNA expression levels are increased in rats treated with paraquat, suggesting a crucial role of IL-10 in lung injury of poisoned rats. In a previous study investigating sucralfate intervention for paraquat poisoning, IL-10 was shown to be decreased by sucralfate treatment in rats treated with paraquat, and both lung and kidney injuries of the rats were improved (14). The role of miRNAs in regulating IL-10 has been widely studied (15,16). A previous study showed the protective mechanism of miR-98 in hepatocellular carcinoma (HCC) and IL-10 was found to be the target gene of miR-98, therefore miR-98 may suppress the progression of HCC by targeting IL-10 (14). Additionally, miR-27b-3p and miR-340-5p were reported to target the 3′untranslated regions (3′UTR) of IL-10 (17). Transfection and transduction assays showed that upregulation of miR-27b-3p and miR-340-5p downregulated IL-10 expression levels (17). Xie et al (18) reported that miR-27a can regulate the inflammatory response of macrophages by targeting IL-10, and the downregulation of miR-27a was considered to be part of a negative-regulatory mechanism to prevent the inflammatory response. In the present study, to investigate the regulatory mechanism of IL-10, the potential miRNA factors regulating IL-10 were predicted using bioinformatics prediction software and databases including miRanda, TargetScan, PiTa, RNAhybrid and PicTar. The present bioinformatic results suggested that miR-27a was a potential regulator for IL-10. The present results suggested that miR-27a may play a role in MODS induced by paraquat poisoning via the regulation of IL-10.

In the present study, the serum expression levels of miR-27a and IL-10 in patients with paraquat poisoning induced MODS were measured and the clinical significance of miR-27a was investigated.

Materials and methods

Patients and sample collection

A total of 82 patients with MODS induced by acute paraquat poisoning were enrolled and were diagnosed according to the diagnostic criteria reported by Fry (19). Patients were admitted to the Emergency Department of The Affiliated Hospital of Weifang Medical University from June 2011 to December 2016. All patients arrived at the hospital within 24 h after poisoning; the poison was ingested orally with an estimated volume of 10–80 ml. In total, 10–15 ml venous blood was collected from all patients on admission to hospital, and the serum was isolated and stored at −80°C for further analysis. The patients (age, 17–52 years; mean age, 30.52±6.93; 40 males and 42 females) did not suffer from immune or immune-related diseases such as diabetes, tumors, chronic liver disease, chronic kidney disease and connective tissue disease. All patients were treated based on a standard treatment protocol including gastric lavage, catharsis, hemoperfusion, antioxidants, high-dose intravenous methylprednisolone and cyclophosphamide (20). A further 88 (age, 18–49 years; mean age, 31.31±7.47; 41 males and 47 females) venous blood samples (5 ml) were collected from healthy fasting participants who had healthy examination results at the same hospital, and were used as the control group. The present study was designed under the review and approval of The Ethics Committee of The Affiliated Hospital of Weifang Medical University. Written informed consent was collected from each participant.

Data collection

All data were recorded in a standard collection for each patient within 24 h of admission, and included: i) Demographic parameters such as age and gender; ii) time interval from paraquat ingestion to admittance in the emergency department; iii) estimated ingestion amount of paraquat; iv) blood biochemical indexes including white blood count (WBC), blood urea nitrogen (BUN), alanine transaminase (ALT) and aspartate transaminase (AST); and v) arterial partial pressure of carbon dioxide (PaCO2) and blood lactic acid (Lac), PaCO2 and Lac were measured using i-STAT blood gas analyzer (Abbott Laboratories).

RNA extraction and reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from serum samples using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Reverse transcription was performed using a miScript RT kit (Qiagen GmbH), and the conditions were 37°C for 15 min and 98°C for 5 min. SYBR green I Master Mix kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used for RT-qPCR to investigate miR-27a expression levels. The following thermocycling conditions were used for the PCR: Initial denaturation at 95°C for 5 min; 30 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 20 sec; and a final extension at 72°C for 10 min. U6 was used as an internal reference for data normalization of miR-27a expression levels. The relative expression levels of miR-27a were determined in relation to U6 using the 2−ΔΔCq method (21). The primers used were as follows: miR-27a forward, 5′-GGGTTCACAGTGGCTA-3′ and reverse, 5′-CTCAACTGGTGTCGTGGA-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′.

Bioinformatics analysis

To investigate the regulatory mechanism of IL-10, the potential miRNA factors regulating IL-10 were predicted using bioinformatics prediction software, miRanda (http://www.microrna.org/microrna/home.do; date of access: August 2010; Memorial Sloan-Kettering Cancer Center), TargetScan (www.targetscan.org/vert_71/; version 7.1), PiTa (http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html; version 6; Weizmann Institute of Science), RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/; accessed September 2017; Bielefeld BioInformatics Service) and PicTar (http://pictar.mdc-berlin.de/; accessed March, 2007). miRanda was used according to the following parameters: Energy value ≤-14 kcal/mol, score ≥80 (22). TargetScan, predictions were ranked according to the predictive efficacy of the target, using context++ scores of the binding sites (23). PiTa was implemented using high-stringency parameters of 7–8 mers and setting the energy value ≤-10 kcal/mol as a general cut-off value (24). RNAhybrid was performed setting the energy value ≤-20 kcal/mol (25). Prediction using PicTar was based on the PicTar score and free energy (26). The bioinformatics results suggested that miR-27a was a potential regulator for IL-10 (Fig. 1).

Figure 1. miR-27a is a potential regulator of IL-10. Schematic illustration of the miR-27a targeting site at the 3′UTR of IL-10 gene. miR-27a, microRNA-27a; 3′UTR, 3′untranslated region; IL-10, interleukin-10; WT, wild-type; Mut, mutant.

ELISA for IL-10

Serum expression level of IL-10 was measured by ELISA using IBL IL-10 ELISA kit (cat. no. 30147233; IBL International GmBH) according to the manufacturer's instructions. The concentration of IL-10 was calculated based on standard curves provided with the kits and results are presented in ng/l.

Statistical analysis

Data are presented as the mean ± SD. The measurement data was analyzed using Student's t test and the categorical data were compared using Chi-squared test. The correlation between IL-10 and miR-27a expression levels was assessed using Spearman's correlation coefficient. A receiver operating characteristic (ROC) curve was used to calculate the specificity and sensitivity of miR-27a expression level, and to assess its feasibility to diagnose MODS induced by paraquat poisoning. Multivariate Cox regression analyses was performed to examine the association between miR-27a expression level and mortality rate. All data analysis was conducted using GraphPad Prism 5.0 software (GraphPad Software, Inc.) and SPSS version 18.0 software (SPSS, Inc.) P<0.05 was considered to indicate a statistically significant difference.

Results

Expression level of miR-27a in patients with paraquat poisoning-induced MODS

The serum miR-27a expression levels in all participants were measured using RT-qPCR. The present results suggested that the serum miR-27a expression level was significantly decreased in patients with MODS compared with the healthy controls (Fig. 2; P<0.001).

Figure 2. Expression level of miR-27a in patients with paraquat poisoning-induced MODS. Comparison of serum expression level of miR-27a in different groups. ***P<0.001 vs. the control group. miR-27a, microRNA-27a; MODS, multiple organ dysfunction syndrome.

Association of miR-27a expression level with the clinicopathological characteristics of patients with MODS induced by paraquat poisoning

To investigate the clinical role of miR-27a, the correlation between miR-27a expression level and the clinicopathological characteristics of all patients was investigated. All patients were classified into low expression group (n=47) and high expression group (n=35) based on the mean value of serum miR-27a expression level (Table I). The present results suggested that miR-27a expression level was closely associated with BUN (P=0.029), PaCO2 (P=0.024), Lac (P=0.012) and APACHE II (27) (P=0.001). However, other clinicopathological parameters showed no significant association with miR-27a expression level including sex, age, time to hospital, estimated amount of paraquat, WBC, BUN, ALT and AST (Table I).

Table I. Association of miR-27a with the clinicopathological characteristics of patients with multiple organ dysfunction syndrome induced by paraquat poisoning.

Table I.

Association of miR-27a with the clinicopathological characteristics of patients with multiple organ dysfunction syndrome induced by paraquat poisoning.

Variable	Low miR-27a expression (n=47)	High miR-27a expression (n=35)	P-value
Sex			0.632
Male	24	16
Female	23	19
Age, years	29.45±6.98	31.97±6.68	0.103
Time elapsed before admission to hospital, h	11.04±4.88	11.11±5.31	0.950
Estimated volume paraquat ingested, ml	37.43±17.81	34.77±19.35	0.522
WBC	15.45±1.55	15.39±1.19	0.834
BUN	12.03±1.80	11.24±1.28	0.029
ALT	152.45±21.08	150.57±26.26	0.721
AST	143.83±21.13	137.94±16.97	0.166
PaCO2	26.59±2.64	27.99±2.87	0.024
Lac	2.72±0.54	2.41±0.55	0.012
APACHE II	9.98±2.34	8.13±2.21	0.001

[i] miR-27a, microRNA-27a; WBC, white blood count; BUN, blood urea nitrogen; ALT, alanine transaminase; AST, aspartate transaminase; PaCO₂, partial pressure of carbon dioxide; Lac, arterial blood lactic acid; APACHE II, the acute physiology and chronic health evaluation II score.

ROC analysis of the diagnostic value of serum miR-27a expression level for MODS induced by paraquat poisoning

A ROC curve is a graphical representation that reflects the correlation between sensitivity and specificity of a laboratory test (28). Based on the serum miR-27a expression levels in patients and controls groups in the present study, a ROC curve was generated to assess the diagnostic value of serum miR-27a expression level for MODS induced by paraquat poisoning. The area under the curve for miR-27a was 0.946, with a sensitivity of 86.6% and specificity of 87.5% at the cutoff value of 2.10 (Fig. 3).

Figure 3. ROC analysis of the diagnostic value of serum miR-27a expression level for multiple organ dysfunction syndrome induced by paraquat poisoning. Diagnostic value of miR-27a using ROC, which had an AUC of 0.946 with a sensitivity of 86.6% and specificity of 87.5%. ROC, receiver operating characteristic; AUC, area under the curve; miR-27a, microRNA-27a.

Association of serum miR-27a expression level with mortality rate

All 82 patients with MODS induced by paraquat poisoning were divided into survival group (n=28) and non-survival group (n=54) based on their status after 30 days from admission. The non-survival group had significantly higher levels of WBC, BUN, ALT, AST and Lac, and lower levels of PaCO2 compared with the survival group (Table II; P<0.001). Additionally, the comparison of traditional score between the survival and non-survival groups showed that non-survivors had a significantly higher APACHE II score (P<0.001). Moreover, miR-27a expression level was significantly different between the survival and non-survival group (P<0.01), with the non-survival group having more patients with low miR-27a expression levels.

Table II. Comparisons between survivors and non-survivors of patients with multiple organ dysfunction syndrome induced by paraquat poisoning.

Table II.

Comparisons between survivors and non-survivors of patients with multiple organ dysfunction syndrome induced by paraquat poisoning.

Variable	Survivors (n=28)	Non-survivors (n=54)	P-value
miR-27a			0.004
Low expression	10	37
High expression	18	17
Sex			0.759
Male	13	27
Female	15	27
Age, years	32.00±7.36	29.76±6.63	0.166
Time elapsed before admission to hospital, h	11.46±4.73	10.87±5.21	0.615
Estimated volume paraquat ingested, ml	34.21±17.16	37.37±19.10	0.465
WBC	14.55±1.35	15.88±1.20	<0.001
BUN	10.58±0.89	12.27±1.64	<0.001
ALT	132.93±24.45	161.35±15.62	<0.001
AST	129.18±18.25	147.61±17.25	<0.001
PaCO2	29.13±3.03	26.18±2.09	<0.001
Lac	2.01±0.32	2.89±0.40	<0.001
APACHE II	7.34±2.20	10.14±2.00	<0.001

[i] miR-27a, microRNA-27a; WBC, white blood count; BUN, blood urea nitrogen; ALT, alanine transaminase; AST, aspartate transaminase; PaCO₂, partial pressure of carbon dioxide; Lac, arterial blood lactic acid; APACHE II, the acute physiology and chronic health evaluation II score.

Furthermore, multivariate Cox regression analyses was performed to examine the association between miR-27a expression level and mortality rate. The present results suggested that miR-27a expression level and APACHE II score may be independent prognostic factors to determine overall survival rates (Table III; P<0.01).

Table III. Multivariate Cox regression analysis for miR-27a in patients with multiple organ dysfunction syndrome induced by paraquat poisoning.

Table III.

Multivariate Cox regression analysis for miR-27a in patients with multiple organ dysfunction syndrome induced by paraquat poisoning.

	Multivariate analysis
Variable	HR	95% CI	P-value
miR-27a	7.156	3.329–15.384	<0.001
Age	0.922	0.496–1.713	0.797
Sex	0.620	0.320–1.201	0.157
Time elapsed before admission to hospital, h	1.059	0.560–2.004	0.859
Estimated volume paraquat ingested, ml	1.099	0.594–2.034	0.763
WBC	1.297	0.707–2.381	0.401
BUN	1.292	0.670–2.489	0.444
ALT	1.209	0.602–2.429	0.595
AST	1.385	0.713–2.689	0.336
PaCO2	0.624	0.199–1.956	0.418
Lac	1.707	0.568–5.125	0.341
APACHE II	2.730	1.282–5.810	0.009

[i] miR-27a, microRNA-27a; WBC, white blood count; BUN, blood urea nitrogen; ALT, alanine transaminase; AST, aspartate transaminase; PaCO₂, partial pressure of carbon dioxide; Lac, arterial blood lactic acid; APACHE II, the acute physiology and chronic health evaluation II score; HR, hazard ratio.

miR-27a expression level is negatively correlated with IL-10 expression level

The bioinformatic results suggested that miR-27a was a potential regulator for IL-10 (Fig. 1). We further investigated the correlation of serum expression levels of miR-27a with IL-10. ELISA was used for the measurement of the serum IL-10 expression levels. The present results suggested that the serum IL-10 expression level was significantly higher in patients with MODS compared with the healthy controls (Fig. 4A; P<0.001).

Figure 4. Correlation between serum expression level of miR-27a and IL-10 in patients with MODS induced by paraquat poisoning. (A) Comparison of serum expression level of IL-10 in different groups. (B) Moderate negative correlation between the serum expression levels of miR-27a and IL-10 in patients with MODS induced by paraquat poisoning, r=−0.5225; P<0.001. ***P<0.001 vs. the control group. miR-27a, microRNA-27a; IL-10, interleukin-10; MODS, multiple organ dysfunction syndrome.

The present study also investigated the correlation between serum expression levels of miR-27a and IL-10 in patients with MODS. The present results identified a moderately negative correlation between the serum expressions level of miR-27a and IL-10 (Fig. 4B; r=−0.5225; P<0.001).

Discussion

Paraquat is a widely used herbicide and has a variety of toxic effects (29). The mechanism of acute paraquat poisoning is complex, with many inflammatory factors and cytokines involved (30). Acute paraquat poisoning can lead to a strong inflammatory reaction (31). Previous studies reported that IL-10 mRNA expression level was increased in paraquat poisoned rats, IL-10 level was decreased by sucralfate treatment and the lung and kidney injuries of the rats were also improved (13,14). Therefore, these previous studies demonstrated the important role of IL-10 in paraquat poisoning. IL-10 inhibits the activity of monocyte macrophages, T cells and natural killer cells (32). IL-10 also inhibits the synthesis and release of other inflammatory cytokines including IL-1β, tumor necrosis factor-α (TNF-α) and IL-8 (32). Systemic inflammatory response syndrome (SIRS) occurs when patients have a severe infection or trauma, and compensated anti-inflammatory response syndrome (CARS) may be subsequently triggered (33). The imbalance of SIRS and CARS damages local tissues cells and affects distant organs, eventually leading to MODS (34). A previous study showed that the incidence of MODS in patients with paraquat poisoning was ~84% (6).

miRNAs are regulatory factors that play crucial roles in regulating growth, maintaining homeostasis of organisms and mediating various pathophysiological processes of diseases, and have become important research topics (35–37). The present bioinformatics results suggested that miR-27a may be a potential regulator for IL-10, thus indicating the potential role of miR-27a in paraquat poisoning. Therefore, a total of 82 patients with MODS induced by paraquat poisoning and 88 healthy controls were enrolled in the present study, and the serum miR-27a expression levels were investigated. The present results suggested the serum miR-27a expression level was significantly lower in patients with paraquat poisoning-induced MODS compared with the healthy controls. The present clinical results suggested that miR-27a may have a significant association with BUN, PaCO2, Lac and APACHE II score. Patients poisoned with paraquat may suffer multiple organ dysfunction of the heart, liver, lungs and kidney (38). BUN is an indicator that reflects kidney function, while PaCO2 and Lac are important indexes reflecting respiratory function and lung injury (39–41). The present study hypothesized that miR-27a may be involved in kidney and lung injury in patients with MODS induced by paraquat poisoning. Ju et al (42) reported that miR-27a can alleviate acute lung injury in mice by inhibiting inflammation. Additionally, miR-27a was shown to inhibit pulmonary fibrosis in rats (43). Aguado-Fraile et al (44) reported that miR-27a was differentially expressed in acute kidney injury (AKI) patients compared with healthy controls, and serum miR-27a expression level can be used as a biomarker to predict AKI predisposition. Thus, previous studies have shown the important role of miR-27a in kidney and lung injury, in line with the present results.

The present study analyzed the diagnostic and prognostic value of miR-27a in patients with paraquat poisoning-induced MODS. The present results suggested that serum miR-27a expression level was decreased in patients with MODS induced by paraquat poisoning. Furthermore, ROC curve analysis suggested that miR-27a may be a sensitive diagnostic biomarker for MODS induced by paraquat poisoning. Previous studies identified that a majority of patients with MODS have lung and kidney injury, and miR-27a was suggested to be involved in organ injury (43,44). Therefore, this may be the possible mechanism for the diagnostic ability of miR-27a in paraquat poisoning. In the present study, according to the overall survival rates after 30 days, patients were divided into survival group and non-survival group. The present results suggested that patients in the non-survival group had higher levels of WBC, BUN, ALT, AST and Lac, and a lower level of PaCO2 compared with the survival group; reflecting the reduction in kidney, liver and lung injury. The present results identified that the non-survival group had lower miR-27a expression levels, suggesting that miR-27a expression may be associated with the mortality of patients with paraquat poisoning-induced MODS. Furthermore, the present multivariate Cox regression analysis results suggested that miR-27a expression level may be an independent prognostic factor to predict mortality. As previously reported, APACHE II score can evaluate the prognosis of acute paraquat poisoning (45). The present results suggested that serum miR-27a expression level may have a significant association with APACHE II score, indicating a significant correlation of miR-27a with the severity of paraquat poisoning. Therefore, the present results suggested serum miR-27a may be a useful clinical tool for predicting the outcome of paraquat poisoning.

As indicated by the present bioinformatics results, miR-27a may be a potential regulator of IL-10. The serum expression level of IL-10 was then investigated, and the present results suggested patients with MODS induced by paraquat poisoning had significantly higher IL-10 expression levels compared with healthy controls. In addition, the present results identified a moderate negative correlation between the serum expression levels of miR-27a and IL-10. In paraquat poisoned rats, IL-10 mRNA expression level is upregulated (13). In addition, early gastrointestinal lavage with sucralfate was found to effectively reduce cytokine levels including IL-10, and to improve lung and kidney injury and survival of paraquat poisoned rats (14). Xie et al (18) showed that miR-27a can regulate the inflammatory response by regulating IL-10, and IL-10 was identified to be the target gene of miR-27a in HEK-293 cells. Therefore, results from previous studies are in line with the present results indicating that serum expression level of IL-10 and miR-27a may reflect the inflammatory response and organ damage in MODS induced by paraquat poisoning. The present results suggested that miR-27a may be involved in the process of paraquat poisoning induced MODS by regulating the inflammatory response. Furthermore, a previous study showed that overexpression of miR-27a decreases STAT3 phosphorylation by downregulating IL-10 expression level, and regulates the activation of the JNK1/STAT3 pathway (18). This may be a possible mechanism for miR-27a in the inflammatory response; however, further study is needed to investigate this mechanism.

The present study has several limitations. The clinicopathological parameters of the enrolled patients is not fully comprehensive as a result of incomplete information collection, and other clinicopathological parameters could have been included, such as serum creatinine and serum cardiac troponin I, for a more thorough examination of the association between miR-27a level and different organ dysfunction. In addition, other inflammatory factors involved in the inflammatory response regulated by IL-10 such as IL-6, TNF-α, interferon and IL-12 should be examined in patients with MODS. This would be helpful for understanding the inflammatory response in MODS induced by paraquat poisoning. Additionally, the present sample size was relatively small, and further studies with larger sample sizes should be performed.

In conclusion, the present results suggested that increased expression level of IL-10 and decreased expression level of miR-27a in serum may reflect the inflammatory response and organ damage in MODS induced by paraquat poisoning. The serum miR-27a expression level may be a potential novel diagnostic and prognostic factor for patients with MODS induced by paraquat poisoning. In addition, miR-27a may be involved in the process of paraquat poisoning induced MODS by regulating the inflammatory response.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

HD made substantial contributions to conception and design, analysis and interpretation of data, draft and revision of the manuscript. HZ and XZ performed the RNA experiments. HW and LW contributed to acquisition of data from patients and samples. All authors were involved in drafting the manuscript and revising it. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was designed under the review and approval of The Ethics Committee of The Affiliated Hospital of Weifang Medical University (ID of ethics approval: 2011-1-2). Written informed consent was collected from each participant.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References