RELATIONSHIP BETWEEN MIR-21 EXPRESSION IN SERUM AND INFLAMMATORY CHANGES AND PROGNOSIS IN SEPTIC RATS
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Acta Medica Mediterranea, 2020, 36: 211
RELATIONSHIP BETWEEN MIR-21 EXPRESSION IN SERUM AND INFLAMMATORY CHANGES
AND PROGNOSIS IN SEPTIC RATS
Yan Zhao1, #, Yadong Yu2, #, Xia Li3, *
1
Department of Emergency, The Affiliated Hospital of Bengbu Medical College & Lianyungang Second People’s Hospital &
Lianyungang Cancer Hospital - 2Department of Emergency, Lian Shui People's Hospital, NO.6 RedSun East Road, Lianshui, Jiangsu,
223400, China - 3Department of Geriatric, Huai’an Hospital Affiliated to Xuzhou Medical University & Huai’an Second People’s
Hospital, 62 Huaihai Road South, Huai’ an 223002, P.R. China
#
These authors contributed equally to this work
ABSTRACT
Objective: To study the relationship between miR-21 expression in serum and inflammatory changes and prognosis in septic rats.
Methods: A rat sepsis model was established by Cecal Ligation and Puncture (CLP). Forty healthy male SD rats were randomly
divided into four groups: a control group (sham operation group, n=10) and an experimental group (n=30), which was further divided
into three groups (CLP operation group, n=10) according to different blood sampling times at 6h, 12h and 24h after operation. Differently
expressed miRNAs in the serum of the control group and rats at 24 hours after CLP operation were screened by fluorogenic quantitative
PCR (qPCR). Levels of miR-21 and inflammatory factors (TNF-α, IL-1 and IL-6) in rat serum were detected at different time points. The
correlation between miR-21 and inflammatory factors was analysed by Spearman correlation, while changes in blood routine indicators
in rats with sepsis at various time points were tested. Lastly, the survival curve of septic rats was drawn using the Kaplan-Meier curve.
Results: Compared with the control group, the expression levels of miR-155 and miR-21 were significantly increased, and miR-
21 was the miRNA with the highest fold difference. The level of miR-21 and inflammatory factors (TNF-α, IL-1 and IL-6) in the sepsis
group was significantly higher than that in the control group (P212 Yan Zhao, Yadong Yu et Al
easily detected in body fluids, such as blood, sweat, sutured layer by layer. The body fluid was lost after
urine, and breast milk(7). By analysing the abnor- intraperitoneal injection of normal saline 30ml/kg.
mal regulation of miRNAs in blood samples from Control group:
patients with sepsis, a large number of studies have (sham operation group) except for not ligating
explored the idea of using extracellular miRNAs as the cecum and cecal perforation, the control group
biomarkers of sepsis(8, 9). Up to now, miR-223, miR- is the same as the CLP group.
146a and miR-150 have been proven to have good
prognosis and diagnostic value for sepsis(10). Blood routine and serum enzyme indexes test
In this study, a rat sepsis model was constructed of rats
by Cecal Ligation and Puncture (CLP). Moreover, Blood routine:
miR-21 was screened by comparing miRNA expres- Take whole blood immediately with anti-co-
sion levels in normal rat serum. The relationship be- agulation with potassium EDTA (EDTA-2K), and
tween the expression of miR-21 and inflammatory then automatically detect it by Coulter-JT automatic
changes and prognosis in septic rats was explored to blood tester.
provide biological indicators for the prediction and Serum enzymology index:
treatment monitoring of sepsis diseases. Whole blood was allowed to stand at 4℃ for
two hours, centrifuged at 3000 rpm/min for 15 min,
Materials and methods and the supernatant was stored in a low temperature
refrigerator at -80℃. Samples were collected and
Experimental animal detected by enzyme-linked immunosorbent assay
SPF healthy male SD rats, aged 4-6 weeks old (ELISA). The specific operation was carried out in
and weighing 200-300 g, were purchased from the strict accordance with the instructions of the ELISA
Nanjing Jingdas Lake Animal Experimental Centre. kit (China, Shanghai), and each sample was repeated
Forty healthy male SD rats were randomly divided three wells and averaged.
into two groups, the control group (sham operation
group, n=10) and the experimental group (CLP sur- Fluorescent quantitation PCR (qPCR)
gery group, n=30). The experimental group was Total RNA was isolated using a Trizol reagent
further divided into three groups according to the (Takara, Dalian), first strand cDNA was obtained
blood sampling points at 6h, 12h, and 24h after sur- using a reverse transcription system kit (Carlsbad,
gery (CLP surgery group, n=10). The gender, age USA), and standard SYBR green PCR kit was
and weight of the four groups of rats were matched used in the StepOne plus system (Applied Biosys-
(P>0.05), which was comparable. All rats were ex- tems) to perform real-time PCR. miR-21 stem loop
perimentally housed for two days to acclimate to the RT primer: 5'-GTCGTATCCAGTGCAGGGTC-
environment and confirmed good growth. The ex- CGAGGTATTCGCACTGGATACGACTCAA-
periment was carried out in strict accordance with CA-3'; sense strand: 5'-GCCCGCTAGCTTAT-
the regulations and methods for the management of CAGACTGATG-3', antisense strand 5'-GTGCA
experimental animals. GGGTCCGAGGT-3'. sense strand: 5'-CATTG-
GGAAAGGTG-CCGAGA-3 ', antisense strand:
Establishment of rat CLP model 5'-ACGCTTAGCCATACAGAGCC-3'; GAPDH,
Sepsis model group: sense strand: 5'-GGGAGCCAAA GG GTCAT-3',
Water fasting was forbidden 12 hours before antisense strand: 5'-GAGTCCTTCCACGATAC-
surgery, and anal temperature was measured before CAA-3'; using TaqMan microRNA reverse tran-
surgery. Abdominal anaesthesia with amobarbital, scription kit and the TaqMan Master Mix II (both
anterior abdominal incision, skin preparation, alco- from Applied Biosystems, USA) tested miR-21 ex-
hol disinfection of abdominal skin, along the ante- pression levels. The fold difference in gene expres-
rior abdominal line for a length of about 2-3 cm in- sion was calculated as 2−ΔΔCt.
cision, free mesenteric and cecum, 4th silk thread in
the cecal vascular arch was ligated at 1-1.5 cm of the Statistical methods
cecal end, and an 18-gauge needle was perforated Statistics were performed using the SPSS 16.0
three times at the end of the cecum. The contents of statistical package (SPSS, Chicago, USA). The
the intestine were squeezed out a little, the alcohol count data were expressed as mean±SD, and the
cotton ball was wiped clean, and the abdomen was index between the two groups was compared usingRelationship between miR-21 expression in serum and inflammatory changes and prognosis in septic rats 213 the Student t-test or the nonparametric Wilcoxon signed rank test. Spearman correlation analysis of miR-21 was used, inflammatory factors, as well as the Kaplan-Meier curve to map the survival curves of septic rats. The significant difference was defined as P
214 Yan Zhao, Yadong Yu et Al the correlation between them were analysed. The Observation on the survival rate of septic rats results show that the expression level of miR-21 Another 50 rats were subjected to CLP surgery was positively correlated with inflammatory fac- to construct a rat model of sepsis. Survival curves of tors TNF-α, IL-6 and IL-1 (P
Relationship between miR-21 expression in serum and inflammatory changes and prognosis in septic rats 215 also provide new insights into effective treatment. caused by sepsis is more severe with time. The lev- According to the report of Lesur et al., a rat model el of HMGB1 in the late inflammatory mediators of sepsis(15, 16) was constructed by the CLP method was significantly increased 24 h after CLP (P
216 Yan Zhao, Yadong Yu et Al
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of changes in coagulation activity induced by antineo- Xia Li
plastic drugs for the treatment of acute myeloid leu- Email: qs28qq@163.com
kemia. Plos One 2017; 12: 175765. (China)You can also read
