Shenling Baizhu Powder Attenuates Lung Injury via The Nrf2/HO-1 Pathway In A Rat Model of Obstructive Sleep Apnea-Hypopnea Syndrome

Summary

This study demonstrates that Shenlingbaizhu powder alleviates OSAHS-induced lung injury in rats. It activates the Nrf2 pathway, which in turn reduces oxidative stress and systemic inflammation in a dose-dependent manner. The protective effects are reversed by the Nrf2 inhibitor ML385, confirming the pathway's crucial role.

Abstract

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is characterized by recurrent upper airway obstruction and chronic intermittent hypoxia, which can promote oxidative stress, inflammation, and lung injury. Here, we established a rat model of OSAHS by exposing animals to chronic intermittent hypoxia in a plexiglass chamber with alternating cycles of nitrogen and compressed air for 8 h per day over 35 days, and used this model to evaluate the protective effects of Shenling Baizhu Powder (SLBZP). Rats were assigned to Control, OSAHS, OSAHS + SLBZP low-, medium-, and high-dose, and OSAHS + SLBZP-H + ML385 groups. Lung histopathology, oxidative stress indices, inflammatory cytokines, and Nrf2/heme oxygenase-1 (HO-1) expression were assessed by hematoxylin and eosin staining, biochemical assays, enzyme-linked immunosorbent assay, quantitative real-time PCR, and western blotting. Compared with the Control group, the OSAHS group showed evident lung injury, reduced Nrf2 and HO-1 expression, decreased superoxide dismutase and glutathione peroxidase activities, increased malondialdehyde levels, and elevated serum inflammatory cytokines. SLBZP alleviated these changes, with greater improvement generally observed at the medium and high doses. Co-treatment with the Nrf2 inhibitor ML385 attenuated the protective effects of SLBZP. These findings indicate that SLBZP alleviates OSAHS-associated lung injury and support the involvement of the Nrf2/HO-1 pathway in its protective effects. This protocol provides a practical experimental framework for evaluating herbal or pharmacologic interventions in OSAHS-related lung injury.

Introduction

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is characterized by recurrent partial or complete collapse of the upper airway during sleep, leading to intermittent hypoxemia, sleep fragmentation, and substantial disease burden^1,2. In addition to its neurocognitive and cardiometabolic consequences, OSAHS has been associated with multiple systemic complications, including pulmonary injury^3,4,5. Chronic intermittent hypoxia (CIH), a central pathophysiological feature of OSAHS, is considered a major driver of oxidative....

Protocol

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Fujian Anburui Biotechnology Co., Ltd (IACUC FJABR; HABR2023091402) and were conducted in compliance with the Guidelines provided by the Management and Utilization of Laboratory Animals.

Animals and grouping

Sixty specific pathogen-free male Wistar rats aged 6–8 weeks and weighing 180–220 g were used in this study. The animals were housed under controlled conditions at 22 ± 2 °C and 50%–60% relative humidity with a 12 h light/dark cycle and free access to standard ch....

Results

SLBZP alleviated lung injury in OSAHS rats, and this effect was attenuated by Nrf2 inhibition (Figure 1)

The Control group showed normal pulmonary architecture, with thin alveolar walls and no obvious inflammatory cell infiltration. In contrast, the OSAHS group exhibited marked lung injury, including disrupted alveolar structure, alveolar epithelial cell damage, interstitial edema, and inflammatory cell infiltration. SLBZP treatment ameliorated these histopathological changes,.......

Discussion

OSAHS is a common chronic disorder characterized by recurrent upper airway obstruction, intermittent hypoxemia, and sleep fragmentation, and it is associated with substantial systemic morbidity^24,25. Increasing evidence indicates that oxidative stress and inflammation are central contributors to OSAHS pathogenesis and its target-organ complications, including pulmonary injury^26,27. In the present study, S.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1% Acid Alcohol	Sinopharm Chemical Reagent Co., Ltd.	https://en.reagent.com.cn/detail.jsp	HE staining differentiation step; verify if prepared in-house
10% Neutral Buffered Formalin	Bioss	C2034	Lung fixation for histology; reasonable match to revised HE method
2×Universal SYBR Green qPCR Supermix	USEVERBRIGHT	S2024L	qPCR
5× Protein Loading Buffer	Beyotime	P0015L	Western blot sample preparation
Absolute Ethanol	Sinopharm Chemical Reagent Co., Ltd.	https://en.reagent.com.cn/enshowproduct.jsp?id=10000418	HE dehydration/rehydration; verify actual concentration series source
Adhesive Slides	CITOTEST	80312-3161	HE
All-in-One First-Strand SuperMix(with dsDNase)	Magen	MD80101	qPCR
Ammonia Water	Sinopharm Chemical Reagent Co., Ltd.	https://en.reagent.com.cn/detail.jsp	HE bluing step; verify actual supplier
Automatic Tissue Processor	Kuohai Medical	KH-TS	HE
Biosafety Cabinet	Shanghai Lishen Scientific Instrument Co., Ltd.	HFsafe-1200LC	ELISA
Centrifuge	Baiyang	B320A	Biochemical Analysis, ELISA
Chemiluminescence Imaging System	E-Blot	Touch Imager XLi	Western blot
Chronic Intermittent Hypoxia Chamber	Custom plexiglass chamber	Custom-built apparatus	CIH/OSAHS model exposure chamber; please verify actual supplier or model
Compressed Air	Local certified gas supplier	Add actual supplier URL	OSAHS model construction; please verify actual supplier
Electronic Analytical Balance	Sartorius	BS224S	Animal Intervention
Electrophoresis Power Supply	Bio-Rad (USA)	POWER PAC 200	Western blot
Embedding Station Cold Plate	Kuohai Medical	KH-BL	HE
Eosin	Epredia	7111	HE
Fluorescence Inverted Microscope	Nikon (Japan)	Ts2-FL	HE
Fully Automated Digital Slide Scanner	Guangzhou Guangying Cell Technology Co., Ltd.	GCell-60	HE
GAPDH Antibody	Proteintech	60004-1-Ig	Western blot
Glutathione Peroxidase (GSH-Px) Assay Kit	Nanjing Jiancheng Bioengineering Institute	A005-1-1	Biochemical Analysis
GraphPad Prism 8	GraphPad Software	https://www.graphpad.com/updates/prism-8-release-notes	Statistical analysis software
Hematoxylin	Epredia	7211	HE
HO-1 Antibody	Proteintech	27282-1-AP	Western blot
HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L)	Proteintech	SA00001-1	Western blot
HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L)	Proteintech	SA00001-2	Western blot
K3 Microplate Reader	Guangdong Danley Technology Co., Ltd.	K3	Biochemical Analysis, ELISA
Liquid Nitrogen	Local certified gas supplier	Add actual supplier URL	Snap-freezing lung tissue; please verify actual supplier
Low-Speed Mini Centrifuge	Hunan Xiangyi Laboratory Instrument Development Co.,Ltd	WTL-4K	Biochemical Analysis, ELISA
Low-Temperature High-Speed Centrifuge	Beckman Coulter, Inc. (USA)	Microfuge-22R Centrifuge	Biochemical Analysis, ELISA
Malondialdehyde (MDA) Assay Kit	Nanjing Jiancheng Bioengineering Institute	A003-1-1	Biochemical Analysis
ML385	MedChemExpress	HY-100523	Nrf2 inhibitor
Neutral Balsam	Solarbio	G8590	Mounting medium for HE-stained sections
Nikon E200 Biological Microscope	Nikon Jiangnan Optical Instruments Co., Ltd. (Nanjing)	E200MV	HE
Nitrogen Gas	Local certified gas supplier	Add actual supplier URL	OSAHS model construction; please verify actual supplier
Nonfat Dry Milk	BD Biosciences	232100	Western blot blocking reagent; verify actual brand if different
Normal Saline (0.9% NaCl)	Shijiazhuang No. 4 Pharmaceutical Co., Ltd.	https://www.cphi-online.com/product/0-9-sodium-chloride-injection/	Vehicle for gavage and tissue homogenization; verify actual supplier
Nrf2 Antibody	Bioss	bs-1074R	Western blot
Nuclease-Free Water	Thermo Scientific	R0582	qPCR reaction preparation; verify actual product if different
Paraffin Wax	Hualing	20220918	HE
Pentobarbital Sodium	Sigma-Aldrich	P3761	Animal anesthesia; verify actual formulation/source if different
Phenylmethylsulfonyl Fluoride (PMSF)	Thermo Scientific	36978	Protease inhibitor used during protein extraction
Pipette	Eppendorf (Germany)	https://www.eppendorf.com/	Biochemical Analysis, ELISA; verify actual model used
Polyvinylidene Fluoride Membrane	Millipore	IPVH00010	Western blot transfer membrane
Protease and Phosphatase Inhibitor Cocktail	Thermo Scientific	78440	Added to RIPA buffer during protein extraction
Protein Quantification Kit (with BSA Standard)	Meilunbio	MA0082-2	Western blot
Rat Interleukin-1 Beta (IL-1β) ELISA Research Kit	Meimian	MM-0047R1	ELISA
Rat Interleukin-6 (IL-6) ELISA Research Kit	Meimian	MM-0190R1	ELISA
Rat Interleukin-8 (IL-8/CXCL8) Detection Kit	Meimian	MM-71113R2	ELISA
Rat Tumor Necrosis Factor Alpha (TNF-α) ELISA Research Kit	Meimian	MM-0180R2	ELISA
Real-Time PCR System	Bio-Rad	1855196	qPCR instrument; reasonable match to revised protocol, verify actual model used
RIPA Lysis and Extraction Buffer	Thermo Scientific	89900	Protein extraction from lung tissue
RNAiso Plus	TaKaRa	9109	qPCR
Rotary Microtome	Thermo Fisher Scientific Instruments Co., Ltd.	HM325	HE
Superoxide Dismutase (SOD) Assay Kit	Nanjing Jiancheng Bioengineering Institute	A001-3	Biochemical Analysis
Tissue Embedding Cassette	CITOTEST	23033	HE
Tissue Embedding Station	Kuohai Medical	KH-BL	HE
Tissue Float Bath and Slide Dryer	Kuohai Medical	KH-P2	HE
Tris-Buffered Saline	Solarbio	T1080	Western blot wash buffer stock; add Tween-20 to prepare TBST
Tween-20	Sigma-Aldrich	P9416	Used to prepare TBST for western blot washes
Ultra-Low Temperature Freezer	Zhongke Meiling Cryogenics Co., Ltd.	DW-HL340	Western blot
Western Chemiluminescent HRP Substrate	Millipore	WBKLS0500	ECL reagent for western blot detection
Xylene	Sinopharm Chemical Reagent Co., Ltd.	10023418	HE deparaffinization and clearing