Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Dec;61(1):666-673.
doi: 10.1080/13880209.2023.2195883.

Ligustrazine inhibits inflammatory response of human endometrial stromal cells through the STAT3/IGF2BP1/RELA axis

Ying Feng  1 , Han Dong  2 , Liyan Zheng  3
Affiliations

Ligustrazine inhibits inflammatory response of human endometrial stromal cells through the STAT3/IGF2BP1/RELA axis

Ying Feng et al. Pharm Biol. 2023 Dec.

Abstract

Context: Endometriosis (EMs) is a gynecological disorder. Ligustrazine has been reported to exert an anti-inflammatory effect on EMs. However, the underlying mechanisms are not completely understood.

Objective: To investigate the effects of ligustrazine on the progression of EMs and the underlying regulatory mechanisms.

Materials and methods: Human endometrial stromal cells (HESCs) were isolated from patients with EMs or control subjects. HESCs were treated with 25, 50, 100, or 200 μM ligustrazine for 1, 3, 6, or 12 h. Western blot and enzyme-linked immunosorbent assays were performed to determine the levels of proteins and inflammatory cytokines, respectively. The binding between STAT3 and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) was assessed by chromatin immunoprecipitation and dual-luciferase reporter assays. The relationship between IGF2BP1 and RELA was assessed by RNA immunoprecipitation and RNA pull-down assay.

Results: Phosphorylated STAT3, IGF2BP1, RELA, TNF-α, IL-6, and IL-1β were upregulated in EMs tissues compared with control tissues (by 1.79-, 2.55-, 1.58-, 3.01-, 2.55-, and 3.34-fold, respectively). Ligustrazine inhibited the expression of p-STAT3, IGF2BP1, RELA, IL-6, TNF-α, and IL-1β. Overexpression of STAT3 promoted RELA-mediated inflammatory responses, while ligustrazine (100 µM) notably reversed this phenomenon. Ligustrazine also alleviated RELA-induced inflammation via downregulating IGF2BP1. STAT3 bound to the promoter of IGF2BP1, and IGF2BP1 bound to the RELA mRNA.

Discussion and conclusion: Ligustrazine inhibited inflammation in EMs via regulating the STAT3/IGF2BP1/RELA axis. These findings propose a new agent against EMs and support the development of ligustrazine-based treatment strategies for EMs.

Keywords: Endometriosis; inflammation; interleukin-1β; interleukin-6; tumor necrosis factor-α.

PubMed Disclaimer

Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
STAT3, IGF2BP1, RELA (p65), IL-6, TNF-α, and IL-1β were upregulated in EMs tissues. (A) The levels of p-STAT3, IGF2BP1, RELA (p65), and STAT3 in normal, EU, and EMs tissues were evaluated by Western blot. β-Actin was used for normalization. (B) The levels of IL-6, IL-1β, and TNF-α in normal, EU, and EMs tissues were detected by RT-qPCR. (C) The levels of IL-6, IL-1β, and TNF-α in normal, EU, and EMs tissues were determined by ELISA. *p < 0.05; **p < 0.01; **p < 0.001.
Figure 2.
Figure 2.
Ligustrazine significantly inhibited the expression of STAT3, IGF2BP1, RELA (p65), IL-6, TNF-α, and IL-1β in HESCs. (A) HESCs were treated with 25, 50, 100, or 200 μM ligustrazine for 6 h. The levels of p-STAT3, STAT3, IGF2BP1, and RELA (p65) in HESCs were measured by Western blot. (B) HESCs were exposed to 100 μM ligustrazine for 1, 3, 6, or 12 h. The levels of p-STAT3, STAT3, IGF2BP1, and RELA (p65) in HESCs were detected by Western blot. (C) HESCs were treated with 25, 50, 100, or 200 μM ligustrazine for 6 h. The concentrations of IL-6, IL-1β, and TNF-α in HESC supernatants were determined by ELISA. (D) HESCs were exposed to 100 μM ligustrazine for 1, 3, 6, or 12 h. The concentrations of IL-6, IL-1β, and TNF-α in HESC supernatants were assessed by ELISA. *p < 0.05; **p < 0.01; **p < 0.001.
Figure 3.
Figure 3.
Ligustrazine inhibited RELA (p65)-mediated inflammation in HESCs by inactivating STAT3. (A) HESCs were transfected with empty vector or pcDNA3.1-STAT3. The level of STAT3 in HESCs was measured by Western blot. (B) HESCs were transfected with empty vector or pcDNA3.1-STAT3, followed by treatment with or without 100 μM Ligustrazine for 6 h. The levels of p-STAT3, IGF2BP1, RELA (p65), and STAT3 in HESCs were assessed by Western blot. (C) The concentrations of IL-6, IL-1β, and TNF-α in HESC supernatants were measured by ELISA. *p < 0.05; **p < 0.01.
Figure 4.
Figure 4.
Ligustrazine alleviated RELA (p65)-mediated inflammation in HESCs by downregulating IGF2BP1. (A) HESCs were transfected with empty vector or pcDNA3.1-IGF2BP1. The level of IGF2BP1 in HESCs was measured by Western blot. (B) HESCs were transfected with empty vector or pcDNA3.1-IGF2BP1, followed by treatment with or without 100 μM ligustrazine for 6 h. The expression of p-STAT3, IGF2BP1, RELA (p65), and STAT3 in HESCs was detected by Western blot. (C) The concentrations of IL-6, IL-1β, and TNF-α in HESC supernatants were measured by ELISA. *p < 0.05; **p < 0.01.
Figure 5.
Figure 5.
STAT3 bound to the promoter of IGF2BP1, and IGF2BP1 bound to the RELA mRNA. (A) HESCs were transfected with shNC or shSTAT3. The levels of STAT3, IGF2BP1, and RELA (p65) in HESCs were assessed by Western blot. (B) AnimalTFDB was used to predict the binding sites between IGF2BP1 and STAT3. (C) ChIP assay detected the enrichment of STAT3 on the IGF2BP1 promoter. (D) The luciferase activity of STAT3 on WT/Mut-IGF2BP1 was examined by dual-luciferase assay. (E) HESCs were transfected with shNC or shIGF2BP1. The levels of IGF2BP1 and RELA (p65) in HESCs were assessed by Western blot. (F, G) The enrichment of IGF2BP1 with the RELA mRNA was assessed by (F) RNA pull-down and (G) RIP assay. (H) The half-life of the RELA mRNA was detected by RT-qPCR. *p < 0.05; **p < 0.01.
Figure 6.
Figure 6.
Ligustrazine attenuated inflammation in EMs via regulating the STAT3/IGF2BP1/RELA axis. HESCs were subjected to the following treatment: 100 μM Ligustrazine, Ligustrazine + pcDNA3.1-STAT3, Ligustrazine + shIGF2BP1, or Ligustrazine + pcDNA3.1-STAT3 + shIGF2BP1. (A) The levels of STAT3, p-STAT3, IGF2BP1, and RELA (p65) in HESCs were measured by Western blot. (B) The concentrations of IL-6, IL-1β, and TNF-α in HESC supernatants were assessed by ELISA. *p < 0.05; **p < 0.01.
See this image and copyright information in PMC

References

    1. Bian Y, Yuan L, Yang X, Weng L, Zhang Y, Bai H, Chen J.. 2021. SMURF1-mediated ubiquitylation of SHP-1 promotes cell proliferation and invasion of endometrial stromal cells in endometriosis. Ann Transl Med. 9(5):362–362. - PMC - PubMed
    1. Chowdhury I, Banerjee S, Driss A, Xu W, Mehrabi S, Nezhat C, Sidell N, Taylor RN, Thompson WE.. 2019. Curcumin attenuates proangiogenic and proinflammatory factors in human eutopic endometrial stromal cells through the NF-κB signaling pathway. J Cell Physiol. 234(5):6298–6312. - PMC - PubMed
    1. Cruz-Zaragoza LD, Dennerlein S, Linden A, Yousefi R, Lavdovskaia E, Aich A, Falk RR, Gomkale R, Schöndorf T, Bohnsack MT, et al. 2021. An in vitro system to silence mitochondrial gene expression. Cell. 184(23):5824–5837.e15. - PubMed
    1. Gao M, Liu L, Zhang X, Li Z, Zhang M.. 2021. Interleukin-6 reverses adriamycin resistance in nasal NK/T-cell lymphoma via downregulation of ABCC4 and inactivation of the JAK2/STAT3/NF-κB/P65 pathway. Environ Toxicol Pharmacol. 85:103639. - PubMed
    1. He Y, Hung SW, Liang B, Zhang R, Gao Y, Chu CY, Zhang T, Xu H, Chung JPW, Wang CC.. 2021. Receptor tyrosine kinase inhibitor sunitinib as novel immunotherapy to inhibit myeloid-derived suppressor cells for treatment of endometriosis. Front Immunol. 12:641206. - PMC - PubMed