Alleviation of Diabetic Tendon Injury via Activation of Tendon Fibroblasts Autophagy under Berberine Treatment

Abstract

Berberine (BBR) is an isoquinoline alkaloid isolated from Coptis chinensis and possesses valuable pharmacological activities, including anti-inflammatory, anti-tumor, and alleviating several complications of type 2 diabetes mellitus (T2DM). However, the role of BBR in regulating diabetic tendon injury remains poorly understood. In this study, a rat model of T2DM was constructed, and cell apoptosis and autophagy were assessed in tendon tissues after BBR treatment through TdT-Mediated dUTP nick-end labeling (TUNEL) assay and immunohistochemical analysis. Tendon fibroblasts were obtained from the rat Achilles tendon, and the role of BBR in regulating cell apoptosis, the production of inflammatory cytokines, and autophagy activation were assessed using flow cytometry, quantitative real-time PCR (qRT-PCR), and western blot analysis. We demonstrated that BBR treatment significantly increased autophagy activation and decreased cell apoptosis in tendon tissues of T2DM rats. In tendon fibroblasts, BBR repressed High glucose (HG)-induced cell apoptosis and production of proinflammatory cytokines. HG treatment resulted in a decrease of autophagy activation in tendon fibroblasts, whereas BBR restored autophagy activation. More important, pharmacological inhibition of autophagy by 3-MA weakened the protective effects of BBR against HG-induced tendon fibroblasts injury. Taken together, the current results demonstrate that BBR helps relieve diabetic tendon injury by activating autophagy of tendon fibroblasts.

Introduction

Diabetes (diabetes mellitus, DM) is a systemic metabolic disorder characterized by hyperglycaemia¹. At present, diabetes has become one of the main diseases threatening human health and life expectancy². More than 90% of cases are type 2 diabetes, a metabolic disease characterized by chronic inflammation³, insulin resistance⁴, and damage to islet β cells⁵, and the prevalence is increasing each year worldwide.

Type 2 diabetes brings a series of serious complications, which have serious effects on the cardiovascular sys....

Protocol

This study was approved by the Research Ethics Committee at the Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine. All animal experiments were approved by the Ethics Committee of the Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine (IACUC number: YYLAC-2019-1). Male Wistar rats (200-240 g, 8 weeks old) were purchased from Shanghai SLAC Laboratory Animal Center.

Discussion

Tendon injury is a common complication in patients with DM²⁷. Tendon fibroblasts play an important role in the wound healing process^28,29. The current study verified that i) BBR increased autophagy activation and decreased cell apoptosis in tendon tissues of DM rats, ii) BBR decreased HG-induced apoptosis of tendon fibroblasts, iii) BBR alleviated HG-induced inflammatory response in tendon fibroblasts, iv) BBR restored autophagy activation.......

Materials

Name	Company	Catalog Number	Comments
1% penicillin/streptomycin	Sigma-Aldrich, St. Louis, MO, USA	516104-M
anti-LC3B	Abcam, CA, USA	ab48394
anti-p62	Abcam	ab91526
anti-β-actin antibody	Abcam	ab8227
Binding buffer	BD Biosciences	556454
DMEM	Thermo Fisher Scientific, Waltham, MA, USA	11965092
EVOS XL Core microscope	Thermo Fisher Scientific	AMEX1000
Goat anti-rabbit H&L HRP-conjugated secondary antibodies	Abcam	ab205718
Leukemiavirus reverse transcriptase	Clontech	639574
Male Wistar rats	Shanghai SLAC Laboratory Animal Co., Ltd		200–240 g, 8 weeks
Oligo (dT)18 Primer	TaKaRa	3806
Primers	Shanghai Sangon Biotech		Synthesized primers for IL-1β, IL-6 and IL-10
RIPA Lysis Buffer	Thermo Fisher Scientific	20-188
RNA extraction kit (Trizol)	TaKaRa	9108Q
StepOne Realtime PCR System	Thermo Fisher Scientific	4376357
TUNEL assay kit	Thermo Fisher Scientific	C10245