Effects of Shikonin on HIF-1α/VEGF Signaling Pathway in Mice with Acute Lung Injury Caused by Sepsis

Summary

The protocol aims to evaluate the effectiveness of shikonin in alleviating acute lung injury caused by sepsis in mice. By targeting the HIF-1α/VEGF pathway, the study investigates how different doses of shikonin influence survival rates, lung pathology, and expression of inflammatory markers, highlighting its potential therapeutic benefits.

Abstract

Sepsis often causes acute lung injury (ALI), a high-mortality complication. The HIF-1α/VEGF pathway plays a key role in sepsis, and shikonin, a natural compound with anti-inflammatory properties, may alleviate lung injury by targeting this pathway. Balb/c mice were randomly divided into four groups: sham group, model group, low-dose treatment group, and high-dose treatment group. The sham group underwent laparotomy without cecal ligation and puncture (CLP), while the model group underwent CLP to induce sepsis-related acute lung injury. After modeling, the low-dose and high-dose treatment groups received shikonin by gavage at doses of 12.5 mg/kg and 50 mg/kg, respectively, once daily for 14 days. The 7-day survival rate of the mice was monitored. Hematoxylin and eosin (HE) staining was used to assess lung tissue pathology, the lung wet/dry (W/D) weight ratio was measured, and a Western blot was performed to detect the expression of HIF-1α, VEGF, TNF-α, and IL-6 in lung tissue. Shikonin significantly improved the survival rate of septic mice, with the greatest effect observed in the high-dose group (p < 0.05). Compared with the model group, the lung W/D ratio and tissue damage in the shikonin-treated groups were significantly reduced in a dose-dependent manner. Additionally, shikonin significantly downregulated the expression of HIF-1α, VEGF, TNF-α, and IL-6, with the high-dose group showing the most pronounced reduction (p < 0.05). Shikonin alleviates acute lung injury in septic mice, potentially by inhibiting the expression of HIF-1α and reducing the production of related inflammatory factors.

Introduction

Sepsis is a severe systemic inflammatory response triggered by infection.¹ It often leads to multiple organ dysfunction and can become life-threatening as the condition progresses.² Despite the use of therapies like mechanical ventilation and supportive care, mortality rates for sepsis-associated ALI remain high, reaching 30%-40%³. These treatments mainly address symptoms rather than the underlying causes, limiting their overall effectiveness. Therefore, identifying new therapeutic approaches that target the causes of lung injury could significantly improve patient outcomes in sepsis.

Protocol

The protocol obtained approval from the Wenzhou Medical University Experimental Animal Center. Male C57Bl/6 mice (5-6 months old; 20–25 g) were used in the current study. The details of the main reagents and the equipment used are listed in the Table of Materials.

1. Preparation of the sepsis mouse model

1. House mice under a constant temperature of 25 °C, 50% humidity, and a 12 h light/dark cycle.
2. After a 7-day acclimatization period, randomly divide the mice into four groups: Sham group, Model group, Low-dose treatment group, and High-dose treatment group, with 20 mice in each group.

Results

To assess the therapeutic potential of shikonin, we first evaluated its effect on survival in septic mice over a 7-day period. Treatment with shikonin improved survival rates in a dose-dependent manner (Figure 1).

Given the widespread occurrence of lung injury in sepsis, we also assessed pulmonary edema in mice. Pulmonary edema was evaluated by measuring the lung wet-to-dry weight (W/D) ratio. The W/D ratio was significantly elevated in the model .......

Discussion

As the gold standard model for sepsis research, the CLP model is a key step in this experiment because CLP mimics the complexity of human sepsis better than many other models. Sepsis-associated ALI remains a critical clinical challenge due to its high mortality rates and the limited effectiveness of current treatments^14,15. Our study demonstrates, for the first time, that shikonin significantly improves survival in septic mice by modulating both HIF-1α and i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
H&E stain kit staining kit	Solaibao Technology	G1120HE
Low-temperature centrifuge	Sigma	3K15
Microplate Reader	Bio-Rad	Model 680
rabbit monoclonal VEGF-receptor1 antibody	Abcam	ab238632
rabbit polyclonal HIF-1α antibody	Abcam	ab51608
SDS-PAGE electrophoresis system	Bio-Rad	MINI Protean2
Shikonin	MedChemExpress	HY-N0822