USF2基因敲低对脓毒症大鼠凝血功能障碍的影响及其机制

doi:10.13481/j.1671-587X.20260118

Abstract

Abstract:

Objective To discuss the effect of upstream transcription factor 2 （USF2） on coagulation dysfunction in the septic rats， and to clarify its potential mechanism based on the protein tyrosine phosphatase non-receptor type 2 （PTPN2）/c-Jun N-terminal kinase （JNK）/sterol regulatory element-binding protein 2 （SREBP2） signaling pathway. Methods Fifteen healthy SD rats were randomly selected from 265 rats as control group （no ligation or puncture）； the remaining 250 rats were used to establish the sepsis models by cecal ligation and puncture （CLP）. Seventy-five successfully modeled rats were randomly divided into model group （CLP）， positive drug group （CLP+20 mg·kg?1 simvastatin）， small interfering RNA （siRNA） negative control （si-NC） group （CLP+transfection with si-NC）， si-USF2 group （CLP+transfection with USF2-siRNA）， and JNK activator group （CLP+transfection with USF2-siRNA+2 mg·kg?1 JNK activator Anisomycin）， with 15 rats in each group. An automatic hematology analyzer was used to detect the platelet （PLT） count of the rats in various groups； an automatic coagulation analyzer was used to detect the prothrombin time （PT）， activated partial thromboplastin time （APTT）， thrombin time （TT）， and the levels of D-dimer （DD） and fibrinogen （FIB） of the rats in various groups； enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of inflammatory factors including interleukin-1β （IL-1β）， interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， C-reactive protein （CRP）， and procalcitonin （PCT） in serum of the rats in various groups； kits were used to detect the superoxide dismutase （SOD） activity and the levels of malondialdehyde （MDA） and glutathione （GSH） in serum of the rats in various groups； HE staining was used to observe the pathomorphology of lung tissue and cecal tissue of the rats in various groups； real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods were used to detect the expression levels of USF2 mRNA and protein and the expression levels of PTPN2， phosphorylated JNK （p-JNK）， JNK， and SREBP2 proteins in lung tissue and cecal tissue of the rats in various groups. Results After 12 d of modeling， the survival rate of the rats in control group was significantly higher than that in model group. Compared with control group， the expression levels of USF2 mRNA and protein in lung tissue and cecal tissue of the rats in model group， positive drug group， si-NC group， si-USF2 group， and JNK activator group were significantly increased （P<0.05）； compared with model group and si-NC group， the expression levels of USF2 mRNA and protein in lung tissue and cecal tissue of the rats in si-USF2 group and JNK activator group were significantly decreased （P<0.05）. Compared with control group， the PLT counts of the rats in model group， positive drug group， si-NC group， si-USF2 group， and JNK activator group were significantly decreased （P<0.05）； compared with model group， the PLT counts of the rats in positive drug group， si-USF2 group， and JNK activator group were significantly increased （P<0.05）； compared with si-NC group， the PLT counts of the rats in si-USF2 group and JNK activator group were significantly increased （P<0.05）； compared with si-USF2 group， the PLT count of the rats in JNK activator group was significantly decreased （P<0.05）. Compared with control group， the APTT， PT， TT and DD levels of the rats in model group， positive drug group， si-NC group， si-USF2 group， and JNK activator group were significantly increased （P<0.05）， and the FIB level was significantly decreased （P<0.05）； compared with model group， the APTT， PT， TT and DD levels of the rats in positive drug group， si-USF2 group， and JNK activator group were significantly decreased （P<0.05）， and the FIB level was significantly increased （P<0.05）； compared with si-NC group， the APTT， PT， TT and DD levels of the rats in si-USF2 group and JNK activator group were significantly decreased （P<0.05）， and the FIB level was significantly increased （P<0.05）； compared with si-USF2 group， the APTT， PT， TT and DD levels of the rats in JNK activator group were significantly increased （P<0.05）， and the FIB level was significantly decreased （P<0.05）. Compared with control group， the levels of IL-1β， IL-6， TNF-α， CRP， PCT and MDA in serum of the rats in model group， positive drug group， si-NC group， si-USF2 group， and JNK activator group were significantly increased （P<0.05）， and the SOD activity and GSH level were significantly decreased （P<0.05）； compared with model group， the levels of IL-1β， IL-6， TNF-α， CRP， PCT and MDA in serum of the rats in positive drug group， si-USF2 group， and JNK activator group were significantly decreased （P<0.05）， and the SOD activity and GSH level were significantly increased （P<0.05）； compared with si-NC group， the levels of IL-1β， IL-6， TNF-α， CRP， PCT and MDA in serum of the rats in si-USF2 group and JNK activator group were significantly decreased （P<0.05）， and the SOD activity and GSH level were significantly increased （P<0.05）； compared with si-USF2 group， the levels of IL-1β， IL-6， TNF-α， CRP， PCT and MDA in serum of the rats in JNK activator group were significantly increased （P<0.05）， and the SOD activity and GSH level were significantly decreased （P<0.05）. Compared with control group， the alveolar structure of the lung tissue of the rats in model group rats was damaged， the villi of the cecal tissue disappeared， and a large number of inflammatory cells infiltrated； compared with model group， the lung tissue alveolar damage and cecal villi damage of the rats in positive drug group， si-USF2 group， and JNK activator group were alleviated， and inflammatory cell infiltration was reduced； compared with si-NC group， the above pathological changes in the lung and cecal tissues of the rats in si-USF2 group and JNK activator group were significantly alleviated； compared with si-USF2 group， the pathological changes in the lung and cecal tissues of rats in JNK activator group were aggravated.Compared with control group， the expression level of SREBP2 protein and the p-JNK/JNK ratio in lung tissue and cecal tissue of the rats in model group， positive drug group， si-NC group， si-USF2 group， and JNK activator group were significantly increased （P<0.05）， and the expression level of PTPN2 protein was significantly decreased （P<0.05）； compared with model group and si-NC group， the expression level of SREBP2 protein and the p-JNK/JNK ratio in lung tissue and cecal tissue of the rats in positive drug group， si-USF2 group， and JNK activator group were significantly decreased （P<0.05）， and the expression level of PTPN2 protein was significantly increased （P<0.05）； compared with si-NC group， the expression level of SREBP2 protein and the p-JNK/JNK ratio in lung tissue and cecal tissue of the rats in si-USF2 group and JNK activator group were significantly decreased （P<0.05）， and the expression level of PTPN2 protein was significantly increased （P<0.05）； compared with si-USF2 group， the expression level of SREBP2 protein and the p-JNK/JNK ratio in lung tissue and cecal tissue of the rats in JNK activator group were significantly increased （P<0.05）， and the expression level of PTPN2 protein was significantly decreased （P<0.05）. Conclusion Knockdown of USF2 gene can significantly improve the pathomorphology of lung tissue and cecal tissue， alleviate coagulation dysfunction， and reduce the levels of inflammatory factors and oxidative stress in septic rats； its mechanism may be related to the regulation of PTPN2/JNK/SREBP2 signaling pathway.

Key words: Upstream transcription factor 2, Sepsis, Coagulation dysfunction, Protein tyrosine phosphatase non-receptor type 2, c-Jun N-terminal kinase, Sterol regulatory element-binding protein 2

CLC Number:

R631.2

Jingyuan WANG,Fang CHEN,Yancun LIU,Shixin LI,Songtao SHOU. Effect of USF2 knockdown on coagulation dysfunction in septic rats and its mechanism[J].Journal of Jilin University(Medicine Edition), 2026, 52(1): 171-181.

Figures/Tables 10

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References 30

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Gene	Sequence（5'-3'）
USF2	F：ATGGAACCAGAACTCCTCGAGAT
USF2	R：CCTTCTCCGTTCGACTTCATTG
GAPDH	F：GATGGACACATTGGGGTT
GAPDH	R：AAAGCTGTGGCGTGATG

Group	Lung tissue		Appendix tissue
Group	USF2 mRNA	USF2 protein	USF2 mRNA	USF2 protein
Control	0.26±0.02	0.33±0.01	0.32±0.01	0.25±0.03
Model	1.64±0.01^*	1.49±0.03^*	1.52±0.03^*	1.77±0.01^*
Positive drug	1.61±0.03^*	1.48±0.02^*	1.48±0.01^*	1.78±0.01^*
Si-NC	1.60±0.01^*	1.47±0.01^*	1.49±0.02^*	1.80±0.02^*
Si-USF2	0.53±0.01^*△#	0.60±0.03^*△#	0.66±0.02^*△#	0.58±0.01^*△#
JNK activator	0.56±0.02^*△#	0.58±0.02^*△#	0.65±0.01^*△#	0.59±0.01^*△#

Group	PLT（×10⁹L^-1）	APTT（t/s）	PT（t/s）	TT（t/s）	DD [ρ_B/（mg·L^-1）]	FIB [ρ_B/（g·L^-1）]
Control	732.56±0.02	21.25±0.01	11.25±0.01	14.25±0.01	0.21±0.01	5.98±0.02
Model	354.17±0.01^*	35.68±0.02^*	28.47±0.02^*	25.69±0.02^*	1.81±0.02^*	1.25±0.06^*
Positive drug	401.25±0.01^*△	31.58±0.01^*△	25.47±0.02^*△	21.14±0.02^*△	1.54±0.02^*△	1.69±0.05^*△
Si-NC	353.25±0.02^*	35.58±0.02^*	28.45±0.01^*	25.47±0.02^*	1.81±0.02^*	1.24±0.06^*
Si-USF2	698.25±0.09^*△#	24.14±0.03^*△#	15.24±0.01^*△#	17.84±0.02^*△#	0.67±0.03^*△#	4.74±0.02^*△#
JNK activator	547.36±0.08^*△#○	28.26±0.02^*△#○	20.14±0.02^*△#○	20.14±0.01^*△#○	1.12±0.02^*△#○	2.58±0.03^*△#○

Group	IL-1β	IL-6	TNF-α	CRP	PCT
Control	26.51±0.42	32.17±0.75	38.01±0.37	24.58±0.49	20.67±2.52
Model	93.42±1.64^*	83.71±1.41^*	95.14±1.55^*	81.45±1.07^*	37.12±5.81^*
Positive drug	85.07±1.52^*△	76.82±1.03^*△	83.04±1.13^*△	67.51±1.12^*△	26.35±3.63^△
Si-NC	94.73±1.58^*	84.97±1.25^*	94.62±1.23^*	81.78±1.04^*	36.18±6.07^*
Si-USF2	51.58±0.96^*△#	44.76±0.89^*△#	45.28±0.69^*△#	37.34±1.01^*△#	23.40±3.90^△#
JNK activator	70.25±1.01^*△#○	62.90±2.34^*△#○	65.35±1.85^*△#○	52.36±1.27^*△#○	30.06±5.77^*△#○

Group	SOD [λ_B/（U·mL^-1）]	GSH [c_B/（μmol·L^-1）]	MDA [c_B/（nmol·L^-1）]
Control	158.35±0.25	74.58±0.47	15.69±0.24
Modle	25.69±0.35^*	14.25±0.14^*	58.69±0.21^*
Positive drug	30.24±0.49^*△	21.47±0.23^*△	47.58±0.31^*△
Si-NC	25.68±0.34^*	14.23±0.12^*	58.68±0.19^*
Si-USF2	98.74±0.65^*△#	57.48±0.36^*△#	29.87±0.25^*△#
JNK activator	85.47±0.61^*△#○	41.25±0.74^*△#○	41.25±0.34^*△#○

Effect of USF2 knockdown on coagulation dysfunction in septic rats and its mechanism

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Group	Lung tissue			Appendix tissue
Group	PTPN2	p-JNK/JNK	SREBP2	PTPN2	p-JNK/JNK	SREBP2
Control	3.65±0.05	0.54±0.12	0.51±0.04	3.46±0.12	0.15±0.08	0.24±0.06
Model	1.21±0.09^*	2.53±0.03^*	3.02±0.05^*	1.27±0.05^*	1.36±0.06^*	1.30±0.05^*
Positive drug	1.75±0.11^*△	2.19±0.06^*△	2.28±0.05^*△	1.80±0.07^*△	0.98±0.07^*△	1.01±0.03^*△
Si-NC	1.27±0.06^*	2.50±0.06^*	3.06±0.04^*	1.35±0.08^*	1.35±0.06^*	1.28±0.08^*
Si-USF2	2.62±0.04^*△#	1.16±0.08^*△#	1.67±0.07^*△#	3.01±0.06^*△#	0.58±0.04^*△#	0.62±0.06^*△#
JNK activator	1.91±0.13^*△#○	1.75±0.10^*△#○	2.09±0.11^*△#○	2.06±0.05^*△#○	0.88±0.03^*△#○	0.95±0.07^*△#○

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