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, accounting for approximately 22% of the global total, with over 90% being T2DM^3,4,5. In all reported pathogeneses, islet β-cell dysfunction has been announced to be a pivotal mechanism during the process of T2DM^6,7.

Mitochondria, being the center of cell energy metabolism, are suborganelles involved in the basic functions of the islet β-cell⁸. Current studies have shown that the occurrence and development of T2DM are closely related to mitochondrial activity, function, and oxidative stress. β-cell failure caused by mitochondrial dysfunction is the core feature of T2DM⁹. β-cells located in the islets are responsible for synthesizing and secreting insulin, a hormone necessary to regulate blood sugar levels. In the course of T2DM development, abnormal mitochondrial function, fortified reactive oxygen species, and oxidative stress reaction break the mitochondrial dynamic balance of β cells and affect the secretion and metabolism of insulin¹⁰.

Tibetan medicine Jiangtangle is composed of Berberis kansuensis, Curcumaelongae Rhizoma, Malvae Fructus, Tribuli Fructus, Thlaspi arvense L., and Phyllanthus emblica L. (Figure 1). It is a yellow powder with a fragrant smell and bitter taste. It can be used to cure dry mouth, polydipsia, emaciation, polyuria, diabetic foot, itchy skin, and other symptoms caused by diabetes. Existing clinical evidence has shown that Berberis bark alkaloids can effectively relieve T2DM by lowering blood sugar, improving insulin resistance, and reducing inflammation¹¹. Curcumin has the potential to ameliorate T2DM by reducing blood glucose levels, enhancing insulin sensitivity, modulating lipid profiles, and exerting anti-inflammatory and antioxidant effects¹². In addition, Malave Fructus, Tribulifructus, Thlaspi arvense L. and Phyllanthus emblica L. also have an anti-diabetic or anti-oxidant activity^13,14,15. At present, the efficacy, material basis, and mechanism of Tibetan medicine Jiangtangle in the therapy of T2DM are still undisclosed. The composition of Chinese Tibetan medicine is complex, and its prevention and therapy of diseases are based on the theory of drug use in holistic and systematic views. Currently, the pharmacochemistry of Chinese materia medica serum can partly explain the absorption components in the blood and has become an important means to explore the material basis of Tibetan medicines¹⁶. This study initially elucidated the protective effect of Tibetan medicine Jiangtangle on mitochondria in vitro, with a focus on testing the novel hypothesis that Jiangtangle-containing serum can mitigate mitochondrial dysfunction in rat pancreatic islet β-cells (RIN-m5F) under high-glucose stress.

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.

Experimental preparation

The required drugs and reagents were prepared according to the experimental flow chart (see Table of Materials). SPF SD rats (8-10 weeks, 260-300 g) were used in this study. All animals were purchased from a commercial supplier (see Table of Materials). After quarantine, the animals were kept at a constant room temperature with a 12:12 h light-dark cycle and fed a standard rodent diet and water.

Animal treatment and serum collection

All rats that underwent adaptive feeding were administered Tibetan medicine Jiangtangle by gavage for 7 days. The clinical dose was 0.6 g/d, which was converted to a rat dose of 1.26 g/kg¹⁷. After 1 h from the last administration, the rats were anesthetized with isoflurane at 3%-4% for induction and 2% for maintenance. The anesthetized rats were then secured on a surgical table, and the surgical site was disinfected with iodophor. An incision was made along the midline of the abdomen to expose the abdominal aorta. Blood was slowly drawn using a heparinized syringe from the aorta (8 mL) and transferred to a centrifuge tube for centrifugation. The collected blood was centrifuged at 2360 x g for 10 min at 4 °C to separate serum. The serum samples were then aliquoted into cryogenic tubes at 5 mL per tube and stored in an -80 °C medical refrigerator (see Table of Materials) for subsequent analysis.

UPLC-ESI-Q-Orbitrap-MS analysis

Preparation of medicinal material samples: Tibetan medicine Jiangtangle (140.0 mg) was accurately weighed and dissolved in 25 mL of methanol (see Table of Materials), followed by 30 min sonication at 40 kHz, 300 W. The resultant solution was filtered through a 0.22 µm membrane to obtain the test sample.

Serum sample preparation: 100 µL of serum was thoroughly mixed with 400 µL of cold methanol and acetonitrile (v/v, 1:1; see Table of Materials) by vortexing. The mixture was processed by sonication (40 kHz, 300 W) for 1 h in ice bath. The mixture was then incubated at -20 °C for 1 h, and centrifuged at 4 °C for 20 min at 14,000 x g. The supernatants were then harvested and dried under vacuum for UPLC-ESI-Q-Orbitrap-MS analysis.

Chromatographic conditions: The supernatants were measured by a UPLC-ESI-Q-Orbitrap-MS system (see Table of Materials). For LC separation, an ACQUITY UPLC HSS T3 column (2.1 mm x 100 mm, 1.8 µm) was used. The mobile phase consisted of A (0.1% formic acid in water) and B (100% Acetonitrile). The flow rate was 0.3 mL/min, with a specific gradient program of 5%-95% B (0-30 min), 95% B (30-35 min). The injection volume was 5 µL. MS data was acquired in positive and negative ESI modes separately. The HESI source had set conditions for spray voltage, capillary temperature, gas flows, etc. The instrument scanned over m/z 70-1050 Da, with different resolutions for full MS and MS/MS. Raw MS data was processed using MS-DIAL for peak alignment. Composition of Tibetan medicine Jiangtangle was identified by accurate mass and MS/MS data against public databases. Only variables with over 50% non-zero values in at least one group were retained.

Cell culture and treatment

RIN-m5F cells (see Table of Materials) were cultured with Dulbecco's Modified Eagle medium (DMEM), 10% fetal bovine serum, and 1% penicillin/streptomycin in a controlled 5% CO₂ incubator (see Table of Materials) at 37 °C. RIN-m5F cells at logarithmic growth stage were taken, counted after digestion, and inoculated into 96-well plates with 1 x 10⁵ cells per well. Using 10-60 mM glucose, cell induction was done to mimic a high glucose environment of T2DM. RIN-m5F cells were divided into control, model, and 2.5%, 5%, and 10% Jiangtangle-containing serum groups. Cells in the control group were treated with DMEM, and other groups were induced with DMEM containing glucose for 24 h. Control and model groups were added with medium, while each serum group was added with DMEM containing 2.5%, 5%, and 10% Jiangtangle-containing serum, and incubated for 24 h at 37 °C with 5% CO₂.

CCK 8 analysis

Cell viability was measured by CCK 8 (see Table of Materials). RIN-m5F cells were inoculated in 96-well plates and treated with glucose (0, 10, 20, 30, 40, 50, and 60 mM). Then, 100 µL of DMEM with Jiangtangle-containing serum was added to the wells and incubated at 37 °C with 5% CO₂ for 24 h. Each group included 6 replicate wells. After 24 h of incubation with glucose and 24 h of incubation with Jiangtangle-containing serum, 10 µL of CCK 8 solution was added and incubated at 37 °C for another 1.5 h. A full wavelength microplate reader (see Table of Materials) was used to detect absorbance values of each hole at 450 nm¹⁶.

Mitochondrial function analysis

As we previously reported¹⁸, the Seahorse XFe 24 analyzer (see Table of Materials) was used to evaluate real-time ATP production rate and glycolysis rate. RIN-m5F cells were inoculated into Seahorse XFe24 V24 microplates. Cells were seeded into XF24 cell culture plates using a two-step protocol: 100 µL of cell suspension was dispensed into each well, and the plate was left undisturbed for 1 h. Then the plate was transferred to a 37 °C, 5% CO₂ incubator for 4 h to promote initial attachment. Next, 150 µL DMEM was added to each well. After modeling for 24 h and drug administration for 24 h, the detection solution was prepared by adding 1% glucose, 1% pyruvate, and 1% glutamine to Seahorse XF DMEM medium. Then, the cells were washed twice with 1 mL detection solution. For using the real-time ATP rate measurement kit and the glycolysis rate assay kit, 56 µL of oligomycin and 62 µL of rotenone/antimycin A were resuspended and added to sensor cartridge wells A and B, respectively. And the cell culture plate was placed in a CO₂-free cell culture incubator for 60 min. Finally, ATP production rate and glycolysis rate were monitored in real time by an automatic injection system. All experiments were conducted at 37 °C, and the data were analyzed using XF Wave software¹⁹.

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