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 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 progression from normal mucosa to chronic non-atrophic gastritis, atrophic gastritis, intestinal metaplasia (IM), dysplasia (Dys), and eventually carcinoma2. PLGC represents a crucial stage in GC development, and timely intervention and monitoring of PLGC are vital for early GC prevention.
Recent clinical evaluations and experimental research have confirmed that traditional Chinese medicine (TCM) is emerging as one of the most effective therapies for treating PLGC3,4. HZJD decoction is a TCM formula developed based on clinical experience and rooted in the TCM theory of heat and dampness removal. Previous studies have demonstrated the beneficial effects of HZJD decoction in the treatment of PLGC, particularly in relieving clinical symptoms and pathological manifestations5,6. Through network pharmacology-related studies, we identified the active components of HZJD decoction as well as their potential targets for PLGC7. The findings from a previous study indicated that HZJD decoction had the ability to enhance diversity, optimize the community structure, and increase the relative abundance of intestinal flora in PLGC rats8. Moreover, it was verified that HZJD decoction could improve PLGC by regulating intestinal microbiota and its metabolites9. In recent work, we demonstrated that HZJD decoction could regulate the dynamic balance of cell proliferation and apoptosis in PLGC cells by downregulating the expression of lnc 51736810.
An increasing number of studies have confirmed that mitophagy plays an important role in various cancers. Mitophagy selectively eliminates damaged and depolarized mitochondrion and further prevents excessive accumulation of cytotoxic reactive oxygen species (ROS) from the dysfunctional mitochondrion, which in turn inhibits tumorigenesis11. Mitophagy, the selective degradation of damaged or dysfunctional mitochondria, is intricately linked to mitochondrial oxidative phosphorylation (OXPHOS). The impairment or absence of mitophagy can lead to a shift in cellular energy metabolism towards aerobic glycolysis, a phenomenon commonly known as the Warburg effect. The increased production of lactate and ketone bodies, resulting from the Warburg effect, contributes to the construction of a tumor microenvironment that is conducive to cell proliferation12.
This study presents a comprehensive protocol for utilizing HZJD decoction as a therapeutic approach to mitigate the progression of PLGC. Through our evaluation, we observed a significant positive effect of HZJD decoction, particularly in its ability to regulate mitophagy. The study provides valuable insights into the potential molecular mechanisms of HZJD decoction in treating PLGC.
Protocol
Representative Results
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 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….
Divulgations
The authors have nothing to disclose.
Acknowledgements
This project was funded by the Natural Science Foundation of Hebei Province of China (H2020423207).
Materials
| 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 |
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