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 system⁶, the eye⁷, the kidney⁷, and nerves⁸, putting diabetic patients at risk for multiple disabilities and even life-threatening health risks. There have been few studies on the musculoskeletal system, especially on the pathological changes in diabetic tendons. In recent years, the incidence of chronic tendinopathy has increased significantly. Tendons can dynamically regulate their capacity to store and deliver energy^9,10. In tendon tissues, tendon fibroblasts play an important role in modulating tendon adaption and tendon repair after injury^9,11. At present, the function of tendon fibroblasts on tendon injury remains unclear.

As an isoquinoline alkaloid, BBR exerts pharmacological effects in a variety of physiological processes, including lowering blood glucose, lowering lipids, lowering cholesterol, anti-inflammatory effects, antibacterial effects, removing reactive oxygen species, and antagonizing nervous system dysfunction^12,13. BBR can increase the uptake and utilization of glucose in adipose tissue and skeletal muscle cells, upregulate the expression of the insulin receptor in liver and skeletal muscle cells, increase the expression of LDL receptor in the liver, and reduce the levels of cholesterol and sugar in plasma¹⁴. Although BBR possesses valuable pharmacological activities in alleviating several complications of DM^15,16,17, the role of BBR in regulating diabetic tendon injury remains poorly understood.

Autophagy must occur at the baseline level in most tissues to withdraw damaged organelles and provide metabolites to maintain metabolic homeostasis^18,19. Autophagy acts as a crucial role in β-cell health, and impaired autophagy is correlated with β-cell dysfunction and diabetes progression^20,21. Emerging studies have demonstrated that BBR-induced activation of autophagy contributes to improving diabetic nephropathy²². Based on the above findings, we explored whether BBR is helpful in alleviating diabetic tendon injury through regulating autophagy. The current results demonstrated that BBR reduced HG-induced tendon fibroblasts injury and the inflammatory response. HG decreased autophagy activation of tendon fibroblasts, whereas BBR treatment restored autophagy activation and resulted in a subsequent increase of tendon fibroblasts viability and reduction of proinflammatory cytokines.

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.

1. Rat model of T2DM

1. Maintain male Wistar rats (200-240 g, 8 weeks old) in a climate-controlled environment with a 12 h light/dark cycle (20 ± 2 °C and 50%-60% relative humidity). Provide food and water ad libitum during the experimental period.
2. Make efforts to minimize animal suffering, including gentle handling, daily cage cleaning, and monitoring.
3. Randomly assign the rats to 3 groups: the control group (n = 5), DM model group (n = 5), and diabetic model treated with BBR group (n = 5).
4. Establish the rat DM model according to a previous description²³.
   1. Administer the rats with a single intravenous injection of streptozotocin (STZ) dissolved in freshly prepared sodium citrate buffer (w/v: 2%) at a dose of 30 mg/kg. Inject the control group intraperitoneally with an equal volume of citrate sodium citrate buffer without STZ.
   2. Assess the blood glucose using a blood gas analyzer. Use the rats with the indicated blood glucose level (≥16.7 mmol/L, continuously for 10 days) for the T2DM model.
   3. After 1 week, randomly divide the T2DM rats into two groups (n = 5 of each group): untreated rats or rats administered 200 mg/kg/day of BBR by gavage for 4 weeks.

2. Primary tendon fibroblasts

1. Sacrifice the rats under anesthesia through intraperitoneal injection of barbiturate (40 mg/kg) and obtain the Achilles tendon as previously reported²⁴.
2. Isolate tendon fibroblasts from tendon tissues²⁵.
   1. Shredthe tendon tissues manually and place them in DMEM containing 0.2% type II collagenase. Agitate vigorously for 3 h at 37 °C.
   2. Remove the medium by centrifugation and add DMEM containing 10% FBS and 1% penicillin/streptomycin to the digested tissue.
   3. Filter the tendon tissues by a 100 µm strainer, pour the filtered solution into 6-well plates, and maintain the tendon fibroblasts in a humidified incubator at 37 °C with 5% CO₂.

3. Cell viability assay

NOTE: Cell counting kit-8 (CCK-8) assay was used to measure cell viability according to the manufacturer's instructions.

1. After trypsinization, plate the tendon fibroblasts in 96-well plates (4 x 10³ cells/well) and then treat them with different doses of glucose (0, 5, 10, 20, 30, and 50 mM) in the presence or absence of BBR (0, 5, 10, 20, 40, and 80 µM) for 48 h.
   NOTE: Glucose and BBR were dissolved in DMEM.
2. Add CCK-8 solution (10 µL) to each well, and incubate the cells for another 2 h at 37°C.
3. Subsequently, measure the absorbance of each well with a microplate reader at a wavelength of 450 nm.

4. Cell apoptosis analysis

NOTE: A propidium iodide (PI) and annexin V-FITC flow cytometry assay was used to analyze the apoptosis rate of tendon fibroblasts.

1. Seed the tendon fibroblasts (5 x 10⁵ cells) in 6-well plates in DMEM for 24 h.
2. Aspirate and discard DMEM from each well. Treat the cells with fresh DMEM containing HG (30 mM) in the presence or absence of BBR (20 µM) for 24 h.
3. Detach the cells with 0.25% trypsin in 1x PBS, harvest the cells with PBS, and centrifuge at 2000 x g for 5 min.
4. Resuspend the cells in binding buffer (Table of Materials) and stain with 10 µL of FITC-conjugated annexin V and 5 µL of PI in the dark for 15 min at room temperature (RT).
5. Then, analyze the cells by a flow cytometer.

5. Quantitative real-time polymerase chain reaction (qRT-PCR)

1. Harvest the tendon fibroblasts and homogenize using an RNA extraction kit (Table of Materials) according to the manufacturer's recommendations.
2. Perform reverse transcriptional PCR with Moloney's murine leukemia virus reverse transcriptase and Oligo (dT) primers (Table of Materials).
3. Carry out qRT-PCR using SYBR green qPCR Mix on real-time PCR System. The cycle conditions are denaturation at 95 °C for 10 min and 45 cycles at 95 °C for 20 s, 55 °C for 20 s, and 72 °C for 30 s.
   NOTE: For details of the primers for IL-1β, IL-6 and IL-10 refer to the Table of Materials. β-actin was used as the reference gene.
4. Calculate the relative expression level using the 2^-ΔΔCT formula, as previously described²⁶ .

6. Western blot analysis

1. After treatment with HG (30 mM) in the presence or absence of BBR (20 µM) for 24 h, lyse the tendon fibroblasts with RIPA buffer (Table of Materials), and quantify the total protein concentration using the bicinchoninic acid assay.
2. Separate equivalent amounts of proteins (50 µg) from each sample by 10% SDS-PAGE at RT and then transfer to polyvinylidene fluoride (PVDF) membranes at 4 °C for 2 h.
3. Block the membranes in 5% non-fat dried milk in TBST and then incubate overnight at 4 °C with the following primary antibodies: anti-LC3B (1:1500), anti-p62 (1:2000), and anti-β-actin (1:3500) antibody.
4. After being washed with TBST, incubate the membranes with goat anti-rabbit H&L HRP-conjugated secondary antibodies (1:5000) at RT for 1 h.
5. Use an ECL chemiluminescence kit to visualize the specific blots and quantify the autoradiograms by densitometry.

7. Immunohistochemical analysis (IHC)

1. Cut the paraffin-embedded foot tendon tissues into 6 µm-thick sections.
2. After fixing the sections in 4% formalin, incubate the sections with anti-LC3 antibody (1:200) overnight at 4 °C.
3. After washing three times using 10 mM PBS (pH7.4 with Tween 20), incubate all sections with goat anti-rabbit HRP-conjugated secondary antibody (1:1000) for 1 h at 37 °C and stain with DAB and hematoxylin for 60 min at RT.
4. Image the stained slides at 20x magnification using an inverted microscope.

8. TUNEL assay

NOTE: Cell apoptosis in tendon tissue was analyzed using a TUNEL assay kit according to the manufacturer's instructions.

1. Cut the paraffin-embedded foot tendon tissues into 6 µm-thick sections.
2. De-paraffinize the sections in xylene and rehydrate them in a graded series of ethanol.
3. After immersing with 3% hydrogen peroxide at RT, incubate the sections with TUNEL reaction mixture for 1 h at 37 °C.
4. Counterstain the nuclei using DAPI. Observe the stained cells under a fluorescence microscope (20x) and determine the percentage of TUNEL-positive cells.

9. Statistical analysis

1. Use appropriate software applications to perform statistical analysis
   NOTE: Here, the data are presented as the mean ± standard deviation (SD) of three independent experiments. Statistical analyses were performed with SPSS 23.0. Student's t-test was performed to compare differences between groups, and one-way analysis of variance (ANOVA) was performed for multiple group analyses. The difference was statistically significant when p < 0.05.

Results

To evaluate the pharmacological role of BBR in relieving diabetic tendon injury, cell apoptosis and autophagy activation in foot tendon tissues of DM rats were assessed in the presence or absence of BBR. Figure 1A showed that the protein level of LC3 (an autophagy marker) was decreased in tendons tissues of DM rats compared with control rats, whereas BBR treatment significantly restored autophagy activation. In addition, cell apoptosis was elevated in tendons tissues of DM rats compared with...

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