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.

Recent studies have identified hypoxia-inducible factor-1α (HIF-1α) as a critical regulator in the progression of sepsis-induced ALI^4,5. HIF-1α accumulates in lung tissue during sepsis, driving the expression of downstream genes that exacerbate inflammation and tissue damage⁶. This process significantly contributes to the worsening of lung injury. Therefore, targeting the HIF-1α signaling pathway offers a promising approach for mitigating the inflammatory and hypoxic responses seen in sepsis-induced ALI.

In the search for more effective treatments, Traditional Chinese medicine (TCM) has provided valuable insights^7,8. Shikonin is an anthraquinone compound extracted from Lithospermum erythrorhizon⁷. It shows notable anti-inflammatory⁹, antibacterial¹⁰, and anti-tumor effects¹¹. Shikonin can alleviate lung injury induced by lipopolysaccharide (LPS), suggesting its potential therapeutic role in lung conditions¹². At the same time, some studies have also proposed that shikonin may alleviate oxidative damage caused by sepsis by regulating the mononuclear macrophage system, balancing pro-inflammatory and anti-inflammatory responses. However, the mechanisms behind shikonin's protective effects in sepsis-induced ALI are not fully understood. This represents a critical gap in current research.

In this study, we aim to explore the protective effects of shikonin in a mouse model of sepsis-induced ALI using cecal ligation and puncture (CLP). CLP technology plays an important role in sepsis research as it can simulate complex systemic inflammatory responses and multi-organ dysfunction, making it suitable for evaluating the effectiveness of novel treatment strategies.CLP technology is relatively more clinically relevant compared to traditional LPS injection methods: it simulates the systemic inflammatory response caused by gut microbiota translocation, which is closer to the pathological process of clinical sepsis. It can be graded and controlled: by adjusting the ligation length and perforation size, the severity of the disease can be controlled, and it is suitable for the entire study of sepsis. In addition, it can simulate similar dynamic changes in multi-organ dysfunction and cytokine response. By investigating the role of shikonin in modulating the HIF-1α/VEGF signaling pathway, we hope to provide insights into new therapeutic strategies that address the underlying mechanisms of sepsis-related lung injury, potentially improving clinical outcomes.

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.
3. Based on references from a previous study¹³, treat the low-dose group with a concentration of 12.5 mg/kg of purpurin for 14 days, and the high-dose group with a concentration of 50 mg/kg for 14 days. For the sham surgery group, perform laparotomy, cecal traction, reduction, and closure without ligation or puncture, and treat with the same dose of physiological saline for 14 days. The experimental steps for the sepsis group are as follows.
4. After overnight fasting (water was allowed), anesthetize the mice with sodium pentobarbital (40-80 mg/kg) under sterile conditions throughout the process. Apply vet ointment on the eyes to prevent dryness while under anesthesia.
5. Check proper anesthetization as slowing down of respiratory rate, decrease in muscle tension, and no obvious stress response when lightly clamping the lower limbs with hemostatic forceps.
6. Perform disinfection with 75% ethanol, 3x. Make a longitudinal incision of about 1 cm in the middle-left position of the mouse abdomen, and cut open the skin, fascia, and muscles in layers to expose the abdominal cavity.
7. Using blunt-tipped ophthalmic forceps in both hands, gently explore the abdominal cavity, locate and free the mouse's cecum, and ligate it at a distance of about 1 cm from the end of the cecum.
8. Use a 21G needle to puncture and ligate the end of the cecum, gently squeeze out a small amount of intestinal content from the puncture site using blunt-tipped ophthalmic forceps and then retrieve it into the cecum.
9. Perform subcutaneous fluid resuscitation with 1 mL of 0.9% sodium chloride solution. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. We note that the animal that has undergone surgery is not returned to the company of other animals until it is fully recovered.
10. After 7 days of CLP modeling, administer excessive anesthesia for euthanasia.
    NOTE: Due to the possible irritating effects of purpurin on the skin, respiratory tract, and digestive tract, as well as its high toxicity to aquatic organisms, environmental pollution, use personal protection during the experiment.

2. Assessment of animal models

1. Mouse survival observation
   1. Perform animal handling as described above. Record the mortality rate within 1 week after modeling for statistical analysis. Some mice die without sepsis before euthanasia, while CLP model mice have a certain mortality rate (Table 1).
   2. Lung wet-to-dry weight ratio measurement
      1. After 24 h of modeling, anesthetize the mice again as per steps 1.4-1.5.
      2. Make a longitudinal incision of approximately 1 cm on the left side of the mouse's clavicle midline, and remove the skin, fascia, and muscle layers to expose the chest cavity with surgical scissors and forceps.
      3. Remove the left lung and drain the surface fluid. Measure the wet weight and then dry it in an 80 °C oven for 72 h to a constant weight to obtain the dry weight.
      4. Calculate lung water content as:
         ​Lung water content = (Wet weight - Dry weight) / Wet weight x 100%.
   3. Histopathological comparison of lung tissues
      1. At specified time points, collect the left lung as described above in step 2.2. Fix the lung in 10% neutral formaldehyde, embed it in paraffin, and section it at 5 µm thickness.
      2. Dewax the lung in xylene, 2x, for 5-10 min each time. Perform series ethanol rehydration with 100%, 95%, 85%, and 75%, for 3 min per gradient. Soak in distilled water for 2 min.
      3. Stain with approximately 100 mL of hematoxylin solution for 10 min, then rinse with distilled water to remove any floating color.
      4. Add about 70 mL of the differentiation solution and soak for 30 s. Then, soak in tap water 2x, each time for 3-5 min.
      5. Add approximately 100 mL of eosin dye solution dropwise for 2 min. Pour out excess dye solution and quickly dehydrate as described below.
      6. Perform dehydration, transparency, and sealing as described below.
         1. Soak samples in gradient ethanol: 75%, 85%, 95%, and 100% ethanol (I) for 2-3 s each. Soak in 100% ethanol (II) for 1 min, and soak in xylene 2x for 1 minute each time. Using a straw or dropper, drip neutral gum onto the surface of tissue slices and spread it evenly as much as possible to avoid the formation of bubbles. Seal and observe under a microscope at 400x.
      7. Select 10 random fields per lung section. Ask a pathologist to score pathological changes such as alveolar edema, hemorrhage, and neutrophil infiltration from 0 (normal) to 4 (severe damage). Use the total score for evaluation.
   4. Western blotting
      1. Extract nuclear proteins from lung tissue according to the nuclear protein extraction kit instructions, and measure protein concentration using the BCA method as per the manufacturer’s instructions.
      2. Prepare protein loading samples with 50 μg of protein loaded per sample. Prepare SDS-PAGE separating gel (10%) and stacking gel (3%). Perform electrophoresis at 80 V for 45 min, then transfer protein to PVDF membrane at 110 V for 60 min after the samples have entered the separating gel.
      3. Transfer proteins to a membrane and block for 60 min. Incubate with primary antibodies (see Table of Materials, 1:1000) overnight at 4 °C, followed by secondary antibody (see Table of Materials, 1:1000) incubation at 37 °C for 2 h.
      4. Wash the membrane with TBST and use ECL for visualization. In a dark room, add the prepared reagents onto a PVDF membrane and perform shaking and fluorescence imaging. ECL reagents interact with biomolecules on the membrane, producing strong chemiluminescence reactions, which are recorded by fluorescence imaging instruments.
      5. Analyze the grayscale value of protein bands using an image analysis system, Band Quantification Method (For detailed information on the system used for analyzing band densitometry, see NIH Image).
   5. Statistical analysis
      1. Use SPSS 26.0 for statistical analysis. Use one-way ANOVA to compare the means of multiple groups, and LSD-t tests for pairwise comparisons. Consider a p-value < 0.05 as statistically significant.

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