rh-relaxin-2 Attenuates Oxidative Stress and Neuronal Apoptosis via the ERK-nNOS-NO Pathway After Germinal Matrix Hemorrhage in Rats

Academic Background

Germinal Matrix Hemorrhage (GMH) is one of the most common neurological disorders in newborns, particularly in preterm infants, and is a major cause of brain injury. GMH not only triggers acute inflammatory responses but also leads to oxidative stress and neuronal apoptosis, resulting in long-term neurological dysfunction. Oxidative stress and neuronal apoptosis are considered significant drivers of post-GMH sequelae. Therefore, identifying effective antioxidant and anti-apoptotic strategies is crucial for mitigating brain injury following GMH.

Relaxin-2, a member of the insulin-like peptide family, exerts its biological functions by binding to the Relaxin Family Peptide Receptor 1 (RXFP1). Previous studies have shown that Relaxin-2 has antioxidant and anti-apoptotic effects in ischemia-reperfusion injury, but its role in GMH remains unclear. Thus, this study aimed to investigate whether Relaxin-2 could alleviate oxidative stress and neuronal apoptosis after GMH through the ERK-nNOS-NO pathway.

Source of the Paper

This research was conducted by Jun Liu, Yonghua Cai, Khalil Ur Rahman, Qixiong Zhou, and others from the Department of Neurosurgery at Nanfang Hospital, Southern Medical University. The corresponding authors are Guozhong Zhang, Ye Song, and Peng Li. The paper was published in 2025 in the journal Fluids and Barriers of the CNS, with the DOI 10.1186/s12987-024-00616-7.

Research Process and Results

1. Study Design and Experimental Procedure

The study consisted of seven experiments, divided into the following steps:

Experiment 1: Expression and Interaction of Endogenous Relaxin-2 and RXFP1

• Subjects: 36 rats, with brain tissue samples collected at 0, 0.5, 1, 3, 5, and 7 days post-GMH.
• Methods: Western blot to detect changes in Relaxin-2 and RXFP1 expression; double immunofluorescence staining and co-immunoprecipitation (Co-IP) to analyze co-localization and interaction between Relaxin-2 and RXFP1.
• Results: Endogenous Relaxin-2 expression decreased 12 hours post-GMH, while RXFP1 expression increased at 12 hours, peaked on day 1, and gradually declined thereafter. Immunofluorescence and Co-IP confirmed that Relaxin-2 and RXFP1 co-localized and interacted in neurons.

Experiment 2: Effects of rh-relaxin-2 on Oxidative Stress and Neuronal Apoptosis

• Subjects: 18 rats, divided into sham, GMH+Vehicle, and GMH+rh-relaxin-2 groups.
• Methods: Western blot to detect apoptosis markers (Bcl-2, Bax) and oxidative stress marker (Romo1); NO quantification.
• Results: rh-relaxin-2 significantly upregulated Bcl-2 and NO while downregulating Bax and Romo1, demonstrating its antioxidant and anti-apoptotic effects.

Experiment 3: TUNEL and DHE Staining to Assess Neuronal Apoptosis and Oxidative Stress

• Subjects: 36 rats, divided into sham, GMH+Vehicle, and GMH+rh-relaxin-2 groups.
• Methods: TUNEL staining to detect neuronal apoptosis; DHE staining to measure oxidative stress levels.
• Results: rh-relaxin-2 significantly reduced TUNEL-positive neurons and DHE fluorescence intensity, further confirming its anti-apoptotic and antioxidant effects.

Experiment 4: Mechanism of RXFP1 in rh-relaxin-2’s Effects

• Subjects: 30 rats, divided into sham, GMH+Vehicle, GMH+rh-relaxin-2, GMH+sgCtrl+rh-relaxin-2, and GMH+sgRXFP1+rh-relaxin-2 groups.
• Methods: Lentivirus-mediated knockdown of RXFP1; Western blot to detect related protein expression.
• Results: RXFP1 knockdown significantly inhibited the anti-apoptotic and antioxidant effects of rh-relaxin-2, indicating that RXFP1 is a key receptor for rh-relaxin-2’s function.

Experiment 5: Mechanism of the ERK Pathway in rh-relaxin-2’s Effects

• Subjects: 30 rats, divided into sham, GMH+Vehicle, GMH+rh-relaxin-2, GMH+Corn Oil+rh-relaxin-2, and GMH+LY3214996+rh-relaxin-2 groups.
• Methods: ERK inhibitor LY3214996 to block the ERK pathway; Western blot to detect related protein expression.
• Results: The ERK inhibitor significantly reversed the anti-apoptotic and antioxidant effects of rh-relaxin-2, indicating that the ERK pathway plays a critical role in rh-relaxin-2’s function.

Experiment 6: Side Effects of rh-relaxin-2

• Subjects: Two groups of rats, one treated with rh-relaxin-2 and the other untreated.
• Methods: Behavioral tests (open field and T-maze) to assess neurological behavior; liver function (AST, ALT) and kidney function (creatinine) tests.
• Results: No significant differences were observed between the rh-relaxin-2-treated and untreated groups in neurological behavior, body weight, or liver and kidney function, indicating its safety.

Experiment 7: Quantification of NO Levels

• Subjects: Brain tissue samples from Experiments 2, 4, and 5.
• Methods: NO levels measured using the Griess reagent.
• Results: rh-relaxin-2 significantly increased NO levels, further supporting its role through the NO pathway.

2. Key Results and Conclusions

• Key Results:

  1. rh-relaxin-2 significantly alleviates oxidative stress and neuronal apoptosis after GMH through the RXFP1-ERK-nNOS-NO pathway.
  2. RXFP1 and the ERK pathway are critical for rh-relaxin-2’s effects.
  3. rh-relaxin-2 showed no significant side effects in long-term behavioral tests or liver and kidney function assessments.

• Conclusions: This study is the first to demonstrate that rh-relaxin-2 alleviates oxidative stress and neuronal apoptosis after GMH through the RXFP1-ERK-nNOS-NO pathway. This finding provides a new potential therapeutic strategy for GMH, with significant scientific and clinical implications.

Research Highlights

1. Innovation: First to reveal the antioxidant and anti-apoptotic effects of rh-relaxin-2 in GMH and elucidate its mechanism of action.
2. Application Value: rh-relaxin-2, as a potential therapeutic agent, demonstrates good safety and efficacy, with potential for clinical application.
3. Methodological Contribution: Comprehensive validation of rh-relaxin-2’s mechanism through various experimental methods (Western blot, immunofluorescence, TUNEL staining, DHE staining, etc.).

Summary

Through systematic experimental design, this study thoroughly investigated the mechanism of rh-relaxin-2 in GMH, confirming its role in alleviating oxidative stress and neuronal apoptosis via the RXFP1-ERK-nNOS-NO pathway. This finding not only deepens our understanding of the pathological mechanisms of GMH but also provides important theoretical foundations for developing new therapeutic strategies.