Therapeutic Effects of Huazhuojiedu Decoction on Precancerous Lesions of Gastric Cancer by Regulating Mitophagy

Summary

Here, we introduce a protocol that demonstrates the therapeutic efficacy of Huazhuojiedu decoction (HZJD) in alleviating precancerous lesions of gastric cancer using mitophagy regulation.

Abstract

This research aims to explore the therapeutic effect and potential mechanisms of Huazhuojiedu decoction (HZJD) for alleviating precancerous lesions of gastric cancer (PLGC) both in vivo and in vitro. HZJD is a traditional Chinese herbal formula consisting of 11 herbs. Sprague-Dawley (SD) rats were randomly divided into four subgroups: control group, model group, positive drug group, and HZJD group. Hematoxylin-eosin (H&E) staining, high iron diamine-alcian blue (HID-AB) staining, alcian blue-periodic acid Schiff (AB-PAS) staining, immunohistochemistry, immunofluorescence, RT-qPCR, and Western blot assays were performed after 10 weeks of HZJD treatment. In vitro, the cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to detect cell proliferation. RT-qPCR and Western blot assays were performed to evaluate mitophagy levels. The results indicated that HZJD could retard the pathological progression in PLGC rats and reduce PLGC cell proliferation. Treatment with HZJD significantly increased the mRNA and protein expression levels of Sirt3, Foxo3a, Parkin, and LC3 II/I, while decreasing the mRNA and protein expression levels of p62 and Tomm20. HZJD was found to have the ability to reverse the decline in mitophagy activity both in vivo and in vitro. In conclusion, the study assessed the impact of HZJD and provided evidence regarding its potential molecular mechanism.

Introduction

Gastric cancer (GC) remains one of the most common malignant diseases affecting the digestive system worldwide. It is estimated that GC accounts for approximately 6% of all cancers worldwide, ranking 5^th among the most frequently diagnosed cancers and 3^rd among cancer-related deaths¹. GC is widely recognized as a progressive, multi-step biological process. Prior to the onset of GC, the gastric mucosa often undergoes several years of precancerous lesions, referred to as precancerous lesions of gastric cancer (PLGC) stages. The Correa cascade theory is widely accepted and provides an explanation for the sequential progressio....

Protocol

All experimental procedures and animal care were approved by the Hebei University of Traditional Chinese Medicine Institutional Animal Care and Use Committee Guidelines (approval number: DWLL2019031) and were performed in accordance with the ethical guidelines. A total of 90 specific pathogen-free male Sprague-Dawley (SD) rats (age = 6 weeks; weight = 150-180 g; see Table of Materials) were raised at constant temperature (24 °C ± 4 °C) and humidity (50%-60%) under controlled dark/light cyc.......

Discussion

PLGC serves as a key process in the progression from chronic gastritis to GC. TCM has been proven to be a promising treatment and intervention for PLGC in recent years^24,25. In recent years, TCM has emerged as a promising approach for the treatment and intervention of PLGC. This is consistent with previous research¹⁰. The results of pathological examination revealed that HZJD decoction obviously attenuated pathological injury in PLGC rats........

Materials

Name	Company	Catalog Number	Comments
1-Methyl-3-nitro-1-nitrosoguanidine	Shanghai McLean Biochemical Technology Co., Ltd., Shanghai, CHN	R030453
3,3’-diaminobenzidine	Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd., Beijing, CHN	ZLI0919
Alcian blue periodic acid schiff staining reagent kit	Beijing Solarbio Technology Co., Ltd., Beijing, CHN	G1285
Anti-fade fluorescence mounting medium	Beijing Solarbio Technology Co., Ltd., Beijing, CHN	S2110
Bicinchoninic acid protein concentration determination kit	Wuhan Servicebio Technology Co., Ltd., Wuhan, CHN	G2026-1000T
CCK-8 reagent kit	Boster Biological Engineering co., Ltd., Wuhan, CHN	AR1160
Confocal fluorescence microscopy	Leica Instruments Co., Ltd., Weztlar, GER	TCS-SP8SR
COX antibody	Abcam Trading Co., Ltd., Shanghai, CHN	ab202554
DAPI staining solution	Beijing Solarbio Technology Co., Ltd., Beijing, CHN	C0065
DEPC	Wuhan Servicebio Technology Co., Ltd., Wuhan, CHN	G3004
EdU cell proliferation kit	Beyotime Biotechnology Co., Ltd., Shanghai, CHN	C0071S
Electrophoresis solution	Boster Biological Technology co., Ltd., Wuhan, CHN	AR0139
Ethanol	Tianjin Baishi Chemical Industry Co., Ltd., Tianjin, CHN	64-17-5
FBS	Gibco Corporation, Gaithersburg, USA	16000044
Fluorescence microscope	Olympus Corporation, Tokyo, JPN	BH2-RFCA
Foxo3a antibody	Abcam Trading Co., Ltd., Shanghai, CHN	23683
GAPDH antibody	Wuhan Sanying Biology Technology Co., Ltd., Wuhan, CHN	10494-1-AP
Gel preparation kit	Boster Biological Technology co., Ltd., Wuhan, CHN	AR0138
GES-1 cell	Procell Life Science&Technology Co., Ltd., Wuhan, CHN	CL-0563
Goat-anti-mouse IgG	CST Biological Reagents Co., Ltd., Shanghai, CHN	4409
Goat-anti-rabbit IgG	Abcam Trading Co., Ltd., Shanghai, CHN	ab150077
Hematoxylin-eosin staining solution	Zhuhai Beso Biotechnology Co., Ltd., Shenzhen, CHN	BA4027
High iron diamine/alcian blue staining reagent kit	Beijing Solarbio Technology Co., Ltd., Beijing, CHN	G2070
ImageJ	National Institutes of Health, Bethesda, USA
Image-Pro Plus 6.0 software	Media Cybernetics Inc., Maryland, USA
Isopropanol	Tianjin Baishi Chemical Industry Co., Ltd., Tianjin, CHN	67-63-0
LC3 antibody	CST Biological Reagents Co., Ltd., Shanghai, CHN	83506
Loading buffer	Boster Biological Technology co., Ltd., Wuhan, CHN	AR0198
Microplate reader	Rayto Life and Analytical Sciences Co., Ltd., Shenzhen, CHN	RT6100
Microtome	Leica Instruments Co., Ltd., Weztlar, GER	RM2245
Mitophagy kit	Dojindo Laboratories, Kyushu Island, JPN	MD01-10
Neutral balsam	Wuhan Servicebio Technology Co., Ltd., Wuhan, CHN	WG10004160
Optical microscope	Olympus Corporation, Tokyo, JPN	BH2-RFCA
P62 antibody	Abcam Trading Co., Ltd., Shanghai, CHN	ab91526
Paraformaldehyde	Biosharp Life Sciences, Anhui, CHN	BL539A
Parkin antibody	CST Biological Reagents Co., Ltd., Shanghai, CHN	32833
Penicillin–streptomycin	Gibco Corporation, Gaithersburg, USA	15140122
phosphate-buffered saline	Wuhan Servicebio Technology Co., Ltd., Wuhan, CHN	G0002-15
PVDF membrane	Millipore Corporation, Michigan, USA	IPVH00010
Redzol	SBS Genetech Co., Ltd., Beijing, CHN	FTR-50
Reverse transcription reagent kit	Igene Biotechnology Co., Ltd., Guangzhou, CHN	QP057
RIPA Buffer solution	Beijing Solarbio Technology Co., Ltd., Beijing, CHN	R002
Roswell Park Memorial Institute	Gibco Corporation, Gaithersburg, USA	11875093
Silicone needle	Zhongke Life Biotechnology Co., Ltd., Beijing, CHN	TFEP-2
siRNA	Wuhan Genecreate Biological Engineering Co., Ltd. ,Wuhan,CHN
Sirt3 antibody	Abcam Trading Co., Ltd., Shanghai, CHN	189860
Sodium salicylate	Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, CHN	S104176
Sprague-Dawley rat	Beijing Huafukang Biotechnology Co., Ltd., Beijing, CHN	110322210102553975
SYBR quantitative PCR kit	Wuhan Servicebio Technology Co., Ltd., Wuhan, CHN	G3320-15
Tomm20 antibody	Abcam Trading Co., Ltd., Shanghai, CHN	ab186735
Transferring buffer	Boster Biological Technology co., Ltd., Wuhan, CHN	AR0141
Trichloromethane	Shanghai McLean Biochemical Technology Co., Ltd., Shanghai, CHN	821112
Tris-buffered saline with Tween 20	Wuhan Servicebio Technology Co., Ltd., Wuhan, CHN	G0004
VDAC1 antibody	Abcam Trading Co., Ltd., Shanghai, CHN	ab15895
Xylene	Tianjin Baishi Chemical Industry Co., Ltd., Tianjin, CHN	1330-20-7
β-actin antibody	Abcam Trading Co., Ltd., Shanghai, CHN	ab8226