Protective Effects of Serum Containing Tibetan Medicine Jiangtangle on Mitochondrial Function in RIN-m5F Pancreatic β-Cells

Summary

This study showed that the Tibetan medicine Jiangtangle-containing serum could improve mitochondrial dysfunction by enhancing mitochondrial and glycolytic ATP levels.

Abstract

β-cell failure caused by mitochondrial dysfunction is the core feature of type 2 diabetes mellitus (T2DM). Tibetan medicine Jiangtangle, composed of Berberis kansuensis, Curcumaelongae Rhizoma, Malvae Fructus, Tribuli Fructus, Thlaspi arvense L., and Phyllanthus emblica L., can be used to remedy T2DM. However, its effects on mitochondrial function remain unclear. This study aims to clarify the pharmacological effects of Tibetan medicine Jiangtangle on mitochondria. At 7 days after pre-administration of Tibetan medicine Jiangtangle, rat serum was obtained by abdominal aorta blood collection. Rat pancreatic islet β-cells (RIN-m5F) were divided into control, model, and Jiangtangle-containing serum groups. The model and Jiangtangle-containing serum groups were induced for injury with DMEM medium containing 50 mM glucose for 24 h. The control group was treated with DMEM medium containing no glucose for 24 h. Then, medium was added to the control and model groups, while Jiangtangle-containing serum groups were incubated for 24 h with medium containing 2.5%, 5%, and 10% Jiangtangle-containing serum. The results of UPLC-ESI-Q-Orbitrap-MS indicated 90 compounds in Jiangtangle, among which 6 prototypical compounds entering the blood. CCK 8 was used to measure cell viability, Seahorse XFe was employed to evaluate mitochondrial energy metabolism and the effect of Jiangtangle-containing serum on the RIN-m5F cells hyperglycemic model was evaluated. Jiangtangle-containing serum augmented RIN-m5F cell viability, mitochondrial ATP level, glycolytic ATP level, basal glycolysis, compensatory glycolysis, OCR, proton leak, and maximal respiration.

Introduction

Diabetes mellitus is a chronic metabolic disease widely prevalent in the world. Its complex pathogenesis poses a serious threat to human health, being a major public health problem to be solved urgently¹. Diabetes is mainly divided into type 1 diabetes mellitus, type 2 diabetes mellitus (T2DM), gestational diabetes, and specific types of diabetes². With the change of lifestyle and the acceleration of the aging process, the prevalence of diabetes has shown a sharp upward trend in the past few decades. China has the largest diabetic population in the world, with more than 118 million people suffering from diabetes, account....

Protocol

The Administrative Committee of Sichuan Scientist Biological Testing Biotechnology Co., Ltd. has approved the animal protocol (Record number: SYST-2024-019). Male Sprague Dawley (SD) rats, aged 8-10 weeks and weighing 260-300 g, were kept in independent ventilation cages in a controlled environment of 23 ± 2 °C and 55%-60% relative humidity for this study. These specific pathogen-free animals were sourced from a commercial supplier (see Table of Materials). All rats underwent a 1-week period of adaptive feeding to acclimate to the new environment before the start of the experiment, with unrestricted access to water and food.

Results

UPLC-ESI-Q-Orbitrap-MS showed that Tibetan medicine Jiangtangle contained compounds such as phenols, alkaloids, flavonoids, nucleosides, organic acids, amino acids, terpenoids, coumarins, glycosides, tannins, lignans, steroids, and others. (Figure 2, Table 1). SD rats were orally administered 1.26 g/kg of Tibetan medicine Jiangtangle once a day. An overview of the serum gating strategy is presented in Figure 3. T.......

Discussion

T2DM is generally regarded as a chronic metabolic disease with a hidden onset and high prevalence, and is accompanied by many diabetes related complications such as ocular, cardiovascular, and renal dysfunction^20,21,22,23. Its clinical manifestations include elevated blood sugar, polyuria, polydipsia, weight loss, and even blindness, which seriously threaten people's lives and health

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
-80 °C medical refrigerator	Mether	MDF-86H118
0.22 µm aqueous membrane	Jinteng	JTMF0445
0.22 µm needle type microporous filter membrane	Jinteng	JTSFM013001
Acetonitrile	Aladdin	A104440
Agilent OpenLab software	Agilent	Version 2.X
Biosafety cabinet	Airtech	BSC-1004 II A2
Cell counting kit-8	Bioground	BG0025
CelMate CO2 Incubator	Esco Micro Pte Ltd.	CLM-240B-8
Chromatographic column	Agilent	SB-C18
Decolorization shaker	Kylin-Bell	TS-2
Dulbecco's modified eagle medium	Gibco	8121587
Electrophoresis buffer	NCM Biotech	20230801
Formic acid	Aladdin	F298778
Glucose	Chengdu Herbpurify Co., Ltd.	P012-180-180222
GraphPad Prism software	GraphPad software, LLC	Version 9.5.1
High speed refrigerated centrifuge	Thermo Fisher Scientific	Legend Micro 17R
Low-speed centrifuge	Hallon	NH5
Methanol	Aladdin	M116118
Penicillin-streptomycin	NCM Biotech	C125C5
Phosphate buffered saline (1×)	Gibco	8120485
Rat pancreatic islet β-cells	ATCC	CRL-11605
ReadMax 1500 light absorption full wavelength enzyme	Flash	ReadMax 1500
Seahorse XF glycolysis rate assay kit	Agilent	103344-100
Seahorse XF real time ATP rate measurement kit	Agilent	103592-100
Seahorse XFe 24 analyzer	Agilent	XFe24
Trypsin (0.25%, 1×)	HyClone	J210045
UPLC-ESI-Q-Orbitrap-MS analyzer	Thermo Scientific	QE Q Exactive
Vortex mixer	Kylin-Bell	XW-80A