1. Academic Validation
  2. Zhilong Huoxue Tongyu Capsule attenuates hemorrhagic transformation through the let-7f/TLR4 signaling pathway

Zhilong Huoxue Tongyu Capsule attenuates hemorrhagic transformation through the let-7f/TLR4 signaling pathway

  • J Ethnopharmacol. 2023 Apr 18;116521. doi: 10.1016/j.jep.2023.116521.
Lu Geng 1 Li-Zhu Zheng 2 Ya-Fei Kang 3 Chuan-Ling Pan 4 Tao Wang 4 Chen Xie 4 Bo Liang 5 Hui-Ling Liao 6
Affiliations

Affiliations

  • 1 The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, China; Internal Medicine Department One, Wenjiang Traditional Chinese Medicine Hospital of Chengdu, Chengdu, China.
  • 2 The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, China; Traditional Chinese Medicine Hospital of Long Chang City, Neijiang, China.
  • 3 Bazhong Hospital of Traditional Chinese Medicine, Bazhong, China.
  • 4 The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, China; College of Integrated Traditional Chinese and Western Medicine, Southwest Medical University, Luzhou, China.
  • 5 Department of Nephrology, The Key Laboratory for the Prevention and Treatment of Chronic Kidney Disease of Chongqing, Chongqing Clinical Research Center of Kidney and Urology Diseases, Xinqiao Hospital, Army Medical University (Third Military Medical University), Chongqing, China. Electronic address: liangbo2018@126.com.
  • 6 The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, China; College of Integrated Traditional Chinese and Western Medicine, Southwest Medical University, Luzhou, China. Electronic address: liaohl@swmu.edu.cn.
Abstract

Ethnopharmacological relevance: Hemorrhagic transformation after acute ischemic stroke is a life-threatening disease that currently has no effective chemotherapy. Zhilong Huoxue Tongyu Capsule (ZL) is an empirical prescription of traditional Chinese medicine that is used to prevent and treat cardiovascular and cerebrovascular diseases in China. However, only a few studies have addressed the mechanisms of ZL in treating hemorrhagic transformation.

Aim of the study: To evaluate the anti-inflammatory effects of ZL on hemorrhagic transformation model rats and lipopolysaccharide (LPS)-induced RAW264.7 macrophages and to explore the underlying molecular mechanisms.

Materials and methods: Murine RAW264.7 cells were treated with ZL and LPS (1 μg/mL), and cell viability was detected by cell counting kit-8 assay. RT-qPCR was used to detect the expression of inflammatory chemokines, MicroRNA let-7a/e/i/f, toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa-B (NF-κB) p65. The protein expression levels of TLR4, MyD88, NF-κB p65, and Apoptosis related molecules were determined by Western blotting. The Apoptosis rate of RAW264.7 macrophages was detected by Annexin V-FITC/PI double staining. A hemorrhagic transformation model in rats was established by intraperitoneal injection of high glucose solution combined with thread embolization. Then, the model rats were observed behaviourally, pathologically, and molecularly. The gene expression of TLR4, MyD88, and NF-κB p65 was measured by RT-qPCR and used to evaluate the protective effect of ZL against hemorrhagic transformation in rats.

Results: ZL (5, 20, 40 μg/mL) was beneficial in cell proliferation. LPS (1 μg/mL) stimulated the production of inflammatory chemokines and inhibited the production of let-7a/e/i/f, with let-7f being influenced most strongly. Moreover, overexpression of let-7f decreased the gene and protein levels of TLR4, MyD88, and NF-κB p65, downregulated TLR4, and inhibited its transcriptional activity. ZL (5, 20, and 40 μg·mL-1) inhibited the production of TLR4, MyD88, and NF-κB p65 and promoted the production of let-7f in a concentration-dependent manner. Furthermore, the blockade of TLR4 antagonized the promoting effects of TLR4 pathway activation in cell inflammation and Apoptosis by downregulating let-7f. Critically, it was confirmed in vivo and in vitro that ZL upregulated the expression of let-7f and inhibited the gene expression of TLR4, MyD88, and NF-κB p65 to reduce inflammatory cell infiltration, which determined the occurrence of hemorrhagic transformation.

Conclusions: ZL can reduce inflammatory response by upregulating let-7f and subsequently inhibiting the TLR4 signaling pathway, thereby decreasing the occurrence of hemorrhagic transformation.

Keywords

Hemorrhagic transformation; Traditional Chinese medicine; Zhilong Huoxue Tongyu Capsule; let-7f/TLR4 signaling pathway.

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