Effects of overexpression of AT2R on LPS-induced inflammatory responses in AML12 cells
10.19405/j.cnki.issn1000-1492.2023.10.018
- Author:
Changyong Xu
1
;
Shihao Zhang
1
;
Wei Wei
1
Author Information
1. Institute of Clinical Pharmacology，Anhui Medical University， Key Laboratory of Anti-inflammatory and Immune Medicine，Ministry of Education， Anhui Collaborative Innovation Center of Anti-inflammatory and Immune Medicine，Hefei 230032
- Publication Type:Journal Article
- Keywords:
angiotensin Ⅱ type 2 receptor;
interleukin-6;
tumor necrosis factor-α;
lipopolysaccharide;
AML12;
plasmid construction
- From:
Acta Universitatis Medicinalis Anhui
2023;58(10):1712-1718
- CountryChina
- Language:Chinese
-
Abstract:
Objective : Angiotensin Ⅱ Type 2 receptor (AT2R) is a major receptor of angiotensin Ⅱ , which has a protective effect on damage to various tissues and organs．In this study，an overexpression plasmid of AT2R was con- structed to explore the effect of overexpression of AT2R on lipopolysaccharide ( LPS ) -induced inflammatory re- sponse in mouse hepatocytes (AML12 cells) ．
Methods :Mice tissues and organs were used as samples to amplify the gene of interest (AT2R) fragments containing EcoR Ⅰ and Hind Ⅲ restriction sites，and then the final product was obtained by digestion and ligation．The final positive clones were sequenced and identified．The pCMV-Flag-N- AT2R plasmid was transfected into HEK 293T cells，and the expression of Flag protein was detected by the Western blot method after 24 h．AT2R expression on AML12 cells was observed by Western blot and laser confocal microsco- py．AML12 cells were treated differently and divided into control group，LPS treatment group，pCMV-Flag-N-AT2R group and pCMV-Flag-N-AT2R + LPS group，and cell viability was detected by CCK8．Western blot detected PCNA proteins and observed cell proliferation ; Cytoinflammatory factor levels were detected by qPCR ; Western blot detec- ted the expression level of nuclear transcription factor NF-кB (p65) in cells.
Results :The identification and se- quencing results of EcoR I and Hind III double restriction showed that pCMV-Flag-N-AT2R plasmid was successful- ly constructed，and the detection results of the Western blot method showed successful expression of AT2R protein. Laser confocal observed that there were AT2R receptors on AML12 cells，and AT2R recombinant plasmids could be expressed on AML12; Compared with the control group，the viability and proliferation ability of LPS-treated AML12 cells were weakened，while the levels of IL-6 and TNF-α increased，and the expression level of nuclear transcription factor NF-кB (p65) increased．Compared with the LPS group，the viability and proliferation of cells in AML12 cells treated with pCMV-Flag-N-AT2R and LPS were enhanced，while the levels of IL-6 and TNF-α decreased．The ex- pression of the nuclear transcription factor NF-кB ( p65 ) decreased．
Conclusion : AT2R overexpression plasmids were successfully constructed and successfully expressed on AML12 cells，and AT2R could inhibit the inflammatory response of LPS-induced AML12 cells.
- Full text:202407121857125799过表达AT2R对LPS诱导...ML12细胞炎症反应的影响_许昌勇.pdf
