Method Article

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

DOI:

10.3791/65271

⸱

June 30th, 2023

 ,  ,  ,  ,  ,  ,  , 
1Hebei University of Chinese Medicine, 2The First Affiliated Hospital of Hebei University of Chinese Medicine, 3Peking University Third Hospital, 4Beijing University of Chinese Medicine Third Affiliated Hospital

In This Article

Summary

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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

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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

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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 5th among the most frequently diagnosed cancers and 3rd 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....

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Protocol

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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 cycle of 12 h. The rats were acclimated to the new environment for 1 week before starting the experiments.

1. Preparati....

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Results

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MNNG induces PLGC progression in animal model and promotes morphological transformation of GES-1 cells

Upon macroscopic observation, the gastric mucosa of rats in the control group appeared uniformly bright red, smooth, and soft, with the mucosal folds arranged in a linear pattern. In contrast to the rats in control group, the gastric mucosa of the rats in model group exhibited paleness and roughness, and the mucosal plicas were flat or even disappeared (F.......

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Discussion

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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 years24,25. 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........

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Disclosures

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All authors declare that they have no conflict of interest.

Acknowledgements

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This project was funded by the Natural Science Foundation of Hebei Province of China (H2020423207).

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1-Methyl-3-nitro-1-nitrosoguanidineShanghai McLean Biochemical Technology Co., Ltd., Shanghai, CHNR030453
3,3’-diaminobenzidineBeijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd., Beijing, CHNZLI0919
Alcian blue periodic acid schiff staining reagent kitBeijing Solarbio Technology Co., Ltd., Beijing, CHNG1285
Anti-fade fluorescence mounting mediumBeijing Solarbio Technology Co., Ltd., Beijing, CHNS2110
Bicinchoninic acid protein concentration determination kitWuhan Servicebio Technology Co., Ltd., Wuhan, CHNG2026-1000T
CCK-8 reagent kitBoster Biological Engineering co., Ltd., Wuhan, CHNAR1160
Confocal fluorescence microscopyLeica Instruments Co., Ltd., Weztlar, GERTCS-SP8SR
COX figure-materials-1 antibodyAbcam Trading Co., Ltd., Shanghai, CHNab202554
DAPI staining solutionBeijing Solarbio Technology Co., Ltd., Beijing, CHNC0065
DEPCWuhan Servicebio Technology Co., Ltd., Wuhan, CHNG3004
EdU cell proliferation kitBeyotime Biotechnology Co., Ltd., Shanghai, CHNC0071S
Electrophoresis solutionBoster Biological Technology co., Ltd., Wuhan, CHNAR0139
EthanolTianjin Baishi Chemical Industry Co., Ltd., Tianjin, CHN64-17-5
FBSGibco Corporation, Gaithersburg, USA16000044
Fluorescence microscopeOlympus Corporation, Tokyo, JPNBH2-RFCA
Foxo3a antibodyAbcam Trading Co., Ltd., Shanghai, CHN23683
GAPDH antibodyWuhan Sanying Biology Technology Co., Ltd., Wuhan, CHN10494-1-AP
Gel preparation kitBoster Biological Technology co., Ltd., Wuhan, CHNAR0138
GES-1 cellProcell Life Science&Technology Co., Ltd., Wuhan, CHNCL-0563
Goat-anti-mouse IgGCST Biological Reagents Co., Ltd., Shanghai, CHN4409
Goat-anti-rabbit IgGAbcam Trading Co., Ltd., Shanghai, CHNab150077
Hematoxylin-eosin staining solutionZhuhai Beso Biotechnology Co., Ltd., Shenzhen, CHNBA4027
High iron diamine/alcian blue staining reagent kitBeijing Solarbio Technology Co., Ltd., Beijing, CHNG2070
ImageJNational Institutes of Health, Bethesda, USA
Image-Pro Plus 6.0 softwareMedia Cybernetics Inc., Maryland, USA
IsopropanolTianjin Baishi Chemical Industry Co., Ltd., Tianjin, CHN67-63-0
LC3 antibodyCST Biological Reagents Co., Ltd., Shanghai, CHN83506
Loading bufferBoster Biological Technology co., Ltd., Wuhan, CHNAR0198
Microplate readerRayto Life and Analytical Sciences Co., Ltd., Shenzhen, CHNRT6100
MicrotomeLeica Instruments Co., Ltd., Weztlar, GERRM2245
Mitophagy kitDojindo Laboratories, Kyushu Island, JPNMD01-10
Neutral balsamWuhan Servicebio Technology Co., Ltd., Wuhan, CHNWG10004160
Optical microscopeOlympus Corporation, Tokyo, JPNBH2-RFCA
P62 antibodyAbcam Trading Co., Ltd., Shanghai, CHNab91526
ParaformaldehydeBiosharp Life Sciences, Anhui, CHNBL539A
Parkin antibodyCST Biological Reagents Co., Ltd., Shanghai, CHN32833
Penicillin–streptomycinGibco Corporation, Gaithersburg, USA15140122
phosphate-buffered salineWuhan Servicebio Technology Co., Ltd., Wuhan, CHNG0002-15
PVDF membraneMillipore Corporation, Michigan, USAIPVH00010
RedzolSBS Genetech Co., Ltd., Beijing, CHNFTR-50
Reverse transcription reagent kitIgene Biotechnology Co., Ltd., Guangzhou, CHNQP057
RIPA Buffer solutionBeijing Solarbio Technology Co., Ltd., Beijing, CHNR002
Roswell Park Memorial InstituteGibco Corporation, Gaithersburg, USA11875093
Silicone needleZhongke Life Biotechnology Co., Ltd., Beijing, CHNTFEP-2
siRNAWuhan Genecreate Biological Engineering Co., Ltd. ,Wuhan,CHN
Sirt3 antibodyAbcam Trading Co., Ltd., Shanghai, CHN189860
Sodium salicylateShanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, CHNS104176
Sprague-Dawley ratBeijing Huafukang Biotechnology Co., Ltd., Beijing, CHN110322210102553975
SYBR quantitative PCR kitWuhan Servicebio Technology Co., Ltd., Wuhan, CHNG3320-15
Tomm20 antibodyAbcam Trading Co., Ltd., Shanghai, CHNab186735
Transferring bufferBoster Biological Technology co., Ltd., Wuhan, CHNAR0141
TrichloromethaneShanghai McLean Biochemical Technology Co., Ltd., Shanghai, CHN821112
Tris-buffered saline with Tween 20Wuhan Servicebio Technology Co., Ltd., Wuhan, CHNG0004
VDAC1 antibodyAbcam Trading Co., Ltd., Shanghai, CHNab15895
XyleneTianjin Baishi Chemical Industry Co., Ltd., Tianjin, CHN1330-20-7
β-actin antibodyAbcam Trading Co., Ltd., Shanghai, CHNab8226

References

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  1. Bray, F., et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 68 (6), 394-424 (2018).
  2. He, J., et al.

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Tags

Huazhuojiedu DecoctionPrecancerous Gastric LesionsMitophagy RegulationTraditional Chinese MedicineGastric CancerHematoxylin Eosin StainingImmunohistochemistryWestern BlotRT qPCRCell Proliferation
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