Yanmin Bu* and Dezhi Zheng

Department of Orthopedics, Tianjin Hospital, Tianjin University, Hexi, Tianjin 300211, China

*Corresponding Author:
Yanmin Bu
Department of Orthopedics, Tianjin Hospital, Tianjin University, Hexi, Tianjin 300211, China
E-mail: buxingtong@163.com
Date of Received 04 September 2022
Date of Revision 13 June 2023
Date of Acceptance 31 January 2024
Indian J Pharm Sci 2024;86(1):156-162  

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DOI: 10.36468/pharmaceutical-sciences.1261

Abstract

Prior studies have reported that bamboo leaf flavone possess biological and pharmacological activities in mammals. Yet, the effect and possible mechanism of bamboo leaf flavone on interleukin-1 beta-induced chondrocyte damage is still unclear. Methods interleukin-1 beta was used to induce human chondrocytes to establish a cell injury model, followed by exposure to different doses of bamboo leaf flavone. Apoptosis rate was analyzed using flow cytometry. Superoxide dismutase and lactate dehydrogenase activities were assessed using a kit. Interleukin-6 and interleukin-10 levels were determined using enzyme-linked immunosorbent assay. Quantitative reverse transcription-polymerase chain reaction analyzed microRNA-127-5p content. MicroRNA-NC and microRNA127-5p mimics were transfected into chondrocytes, followed by treatment with interleukin-1 beta for 24 h. Cleaved caspase-3 and cleaved caspase-9 protein expression were monitored using Western blot. After bamboo leaf flavone treatment, apoptosis rate, cleaved caspase-3 and cleaved caspase-9 protein levels, lactate dehydrogenase activity, and interleukin-6 level were decreased, while microRNA-127-5p expression, superoxide dismutase activity, and interleukin-10 level were increased in dose-dependent ways. Upregulated microRNA-127-5p hindered apoptosis rate, cleaved caspase-3 and cleaved caspase-9 protein levels, the activity of lactate dehydrogenase, and the level of interleukin-6, and increased superoxide dismutase activity and interleukin-10 level. MicroRNA-127-5p knockdown could antagonize the effects of bamboo leaf flavone on interleukin-1 beta-mediated chondrocyte apoptosis, oxidative stress and inflammation. Bamboo leaf flavone exposure could inhibit cell apoptosis, oxidative stress and inflammation by up-regulating microRNA-127-5p expression, thereby reducing interleukin-1 beta-induced chondrocyte damage.

Keywords

Interleukin-1 beta, human chondrocytes, bamboo leaf flavone, microRNA-127-5p, apoptosis, oxidative stress

As a most prevalent chronic joint disease all over the world, Osteoarthritis (OA) can be responsible for chronic pain, stiffness, and even disability, which is becoming a major public health problem[1,2]. Currently, chronic inflammation, metabolic disorders and matrix degradation of articular cartilage have been considered the primarily characteristics of OA[3,4]. Emerging evidence have indicated that the suppression of chondrocyte apoptosis, oxidative stress, and inflammatory response is beneficial in blocking OA progression[5-7]. However, the pathogenesis of OA has not yet been elucidated. Recently, numerous studies have presented that active components extracted from natural plants have anti-inflammatory and anti-oxidant properties that might hinder OA progression[8,9]. Belong to the phenolic fraction of bamboo leaves, Bamboo Leaf Flavone (BLF) has anti-oxidative stress effects and reduce hippocampal neuronal damage via repressing the inflammatory response in the hippocampus of the brain[10,11]. However, studies on the correlation between BLF and OA have not been reported. Considerable laboratory research has shown that microRNAs (miRNAs), a single-stranded non-coding Ribonuclic Acid (RNA) molecule, can modulate diverse biological processes via hampering the degradation of target genes[12]. Previous reports have described that miR-127-5p might diminish Interleukin-1 Beta (IL-1β)-triggered chondrocyte inflammation and apoptosis[13,14]. Nevertheless, whether miR-127-5p could be a potential target of BLF for OA treatment has not been elucidated. Herein, we focused on whether BLF/miR-127-5p might control IL-1β- challenged chondrocyte injury.

Materials and Methods

Reagents:

BLF and human chondrocytes were respectively provided by RisingPointBio (Wuhan, China) and MingzhouBio (Ningbo, China). Gibco (Grand Island, New York, United States of America (USA)) offered Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), and trypsin. Invitrogen (Carlsbad, California, USA) offered Lipofectamine™ 2000. RiboBio (Guangzhou, China) provided miR-127-5p mimics and anti-miR-127-5p. Besides, Trizol reagent, Reverse transcription and fluorescent quantitative Polymerase Chain Reaction (PCR) reagents were purchase from Thermo Fisher (Grand Island, New York, USA). Cell apoptosis detection kit was acquired from Solarbio (Beijing, China). JianchengBio (Nanjing, China) offered Superoxide Dismutase (SOD) and Lactate Dehydrogenase (LDH) detection kits. Mlbio (Shanghai, China) provided IL-6 and IL-10 detection kits. Amyjet (Wuhan, China) and Abcam (Cambridge, United Kingdom) respectively offered Rabbit anti-human cleaved caspase-3, cleaved caspase-9 antibody and secondary antibodies.

Method:

Cell treatment and transfection: Referring to the previous description[15], 10 ng/ml IL-1β was applied to stimulate chondrocytes in DMEM medium for 24 h, marked as IL-1β group. Synchronously, control group was normal cultured chondrocytes. In 6-well plates, chondrocytes were exposed to 0 μg/ml, 100 μg/ml, and 200 μg/ml BLF[16] in IL-1β- triggered DMEM medium, generating IL-1β+BLFL/ M/H group. Based on Lipofectamine method, we overexpressed miR-127-5p by transfecting miRNC or miR-127-5p mimics into chondrocytes, which were incubated with 10 ng/ml IL-1β- induced DMEM medium, namely IL-1β+miR-NC/ miR-127-5p group. In addition, anti-miR-NC or anti-miR-127-5p transfected chondrocytes were exposed to 200 μg/ml BLF and IL-1β in DMEM medium termed as IL-1β+BLF+anti-miR-NC/antimiR- 127-5p group.

Flow cytometry: Cell apoptosis rate was detected in this experiment. In short, collected chondrocytes were subjected to 0.25 % trypsin digestion and 3000 r/min centrifugation for 6 min. After discarding the supernatant, 5 μl Annexin V-Fluorescein Isothiocyante (FITC) and 5 μl Propidium Iodide (PI) were added into cells, which then were analyzed using FACS Calibur flow cytometer.

Assessment of SOD and LDH activates: After being collected chondrocyte culture supernatant from each group, LDH activity was monitored by the kit. In parallel, Radio-Immunoprecipitation Assay (RIPA) lysate was added into chondrocytes, which were centrifuged. Then, supernatant was aspirated and submitted to detect the activity of SOD using the kit.

Enzyme-Linked Immunosorbent Assay (ELISA): In general, the culture medium of chondrocytes was collected. Then, the secretions of IL-6 and IL-10 were examined using ELISA kits.

Quantitative Reverse transcription (qRT)- PCR: After adding 1 ml of Trizol reagent, total chondrocyte RNA were generated, which then were transcribed to complementary Deoxyribonucleic Acid (cDNA) according to the reverse transcription kit. Subsequently, qRT-PCR amplification reaction system; 2.5 μl 10×PCR buffer, 2.5 μl Magnesium sulfate (MgSO4), 2.5 μl Deoxynucleoside Triphosphate (dNTP), 0.5 μl forward primers, 0.5 μl reverse primers, 2 μl cDNA, 25 μl RNase-free double distilled Water (ddH2O). Procedure includes 95° pre-denaturation for 2 min, 95° denaturation for 60 s, 60° annealing 30 s, annealing at 60° for 60 s, extension at 72° for 30 s, a total of 36 cycles. Finally, U6 acted as the internal reference for miR- 127-5p, and data were analyzed with 2-ΔΔCt method.

Western blot:

In brief, collected chondrocytes were mixed with 500 μl RIPA lysate for total cellular protein. Samples were taken for Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) electrophoresis. Then, separated protein gel was shifted to Polyvinylidene Difluoride (PVDF) membrane, which was added with cleaved caspase-3 (1:1000), cleaved caspase-9 (1:1000), Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) (1:2000). After being added with secondary antibody (1:3000) incubated at 37° for 1 h, the results were analyzed using ImageJ software.

Statistical analysis:

The data in this research was analyzed using Statistical Package for the Social Sciences (SPSS) 21.0 and expressed as (x̄ ±s). Difference were recognized as significant when p<0.05. Comparison was analyzed based on Student’s t-test or one-way Analysis of Variance (ANOVA).

Results and Discussion

Referring to data exhibited in fig. 1 and Table 1, chondrocyte apoptosis rate, cleaved caspase-3 and cleaved caspase-9 protein levels, and LDH activity were improved after IL-1β treatment, but SOD activity was reduced. Meanwhile, BLF exposure might repress apoptosis rate, cleaved caspase-3 and cleaved caspase-9 protein levels, and LDH activity in IL-1β induced chondrocytes with increasing BLF doses, but promote SOD level.

Group Apoptosis rate % Cleaved caspase-3 Cleaved caspase-9 SOD/U·l-1 LDH/U·l-1
Control 6.80±0.51 0.18±0.02 0.25±0.03 309.24±26.74 157.80±15.62
IL-1β 22.40±1.54* 0.75±0.06* 0.87±0.07* 105.55±12.20* 600.66±27.90*
IL-1β+BLF-L 19.95±1.06# 0.61±0.04# 0.72±0.06# 149.96±12.64# 522.32±19.02#
IL-1β+BLF-M 15.28±0.88#& 0.44±0.03#& 0.53±0.04#& 227.20±8.70#& 401.04±21.94#&
IL-1β+BLF-H 11.31±0.67#&@ 0.24±0.02#&@ 0.32±0.02#&@ 261.23±21.62#&@ 281.63±23.76#&@
F 361.745 379.109 270.276 195.894 590.879
p 0.000 0.000 0.000 0.000 0.000

Table 1: BLF Relieved IL-1β Evoked Injury (x̄±s, n=9)

BLF

Fig. 1: BLF treatment repressed IL-1β caused chondrocyte apoptosis, (1): Control; (2): IL-1β; (3): IL-1β+BLF-L; (4): IL-1β+ BLF-M and (5): IL-1β+ BLF-H

As shown in Table 2, IL-6 secretion were apparently enhanced in chondrocyte after IL-1β treatment, but IL-10 level was decreased. Beyond that, BLF might relieve IL-6 content in IL-1β treated chondrocytes in concentration-dependent ways, and boost IL-10 level.

Group IL-6/pg·ml-1 IL-10/pg·ml-1
Control 46.18±6.39 479.38±33.22
IL-1β 226.27±15.53* 125.13±13.52*
IL-1β+BLF-L 191.04±20.27# 200.19±10.98#
IL-1β+BLF-M 146.28±11.73#& 289.53±20.29#&
IL-1β+BLF-H 79.84±6.13#&@ 382.31±18.40#&@
F 294.388 414.591
p 0.000 0.000

Table 2: BLF Hindered IL-1β Induced Inflammatory Factors in Chondrocytes (x̄±s, n=9)

Results from Table 3 showed that miR-127-5p level was obviously reduced by IL-1β treatment. Furthermore, BLF might significantly overturn the repression of IL-1β on miR-127-5p expression in chondrocyte in a dose-dependent manner.

Group miR-127-5p
Control 1.00±0.00
IL-1β 0.16±0.02*
IL-1β+BLF-L 0.32±0.03#
IL-1β+BLF-M 0.57±0.04#&
IL-1β+BLF-H 0.77±0.05#&@
F 948.408
p 0.000

Table 3: Influences of BLF on miR-127-5p Level (x̄±s, n=9)

As displayed in fig. 2 and Table 4, overexpression of miR-127-5p might clearly decline cleaved caspase-3 and cleaved caspase-9 protein level, elevate SOD activity and IL-10 level, and curb LDH activity and IL-6 content in chondrocytes after IL-1β stimulation.

Group miR-127-5p Apoptosis rate % Cleaved caspase-3 Cleaved caspase-9 U·l-1 pg·ml-1
SOD LDH IL-6 IL-10
IL-1β+miR-NC 1.00±0.00 22.61±1.68 0.76±0.07 0.84±0.06 106.18±11.49 603.71±49.31 232.84±19.34 127.75±12.63
IL-1β+miR-127-5p 3.67±0.11* 12.88±0.76* 0.35±0.03* 0.36±0.04* 242.71±11.99* 333.46±19.62* 116.19±9.38* 326.69±25.10*
t 72.818 15.831 16.151 19.969 24.664 15.277 16.281 37.936
p 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

Table 4: Effects of miR-127-5p on Apoptosis (x̄±s, n=9)

miR

Fig. 2: miR-127-5p affected chondrocyte apoptosis, (1): IL-1β+miR-NC and (2): IL-1β+miR-127-5p

As presented in fig. 3 and Table 5, miR-127- 5p deficiency might partly reverse BLFmediated apoptosis and cleaved caspase-3 and cleaved caspase-9 protein levels inhibition, SOD activity promotion, and LDH activity repression in chondrocytes after IL-1β treatment. Downregulation of miR-127-5p might effectively abrogate BLF-triggered IL-6 content repression and IL-10 level promotion in IL-1β-treated chondrocytes (Table 6).

Group miR-127-5p Apoptosis rate % Cleaved caspase-3 Cleaved caspase-9 U·l-1
SOD LDH
IL-1β+BLF+anti-miR-NC 1.00±0.00 11.27±0.84 0.22±0.02 0.31±0.03 265.59±21.51 280.59±30.49
IL-1β+BLF+anti-miR-127-5p 0.30±0.04* 21.22±1.29* 0.68±0.05* 0.75±0.05* 126.32±9.39* 547.16±39.09*
t 52.500 19.391 25.626 22.638 17.802 16.131
p 0.000 0.000 0.000 0.000 0.000 0.000

Table 5: BLF/miR-127-5p Regulated Chondrocyte Damage (x̄±s, n=9)

chondrocytes

Fig. 3: Effect of interfering miR-127-5p on IL-1β-induced apoptosis of chondrocytes after BLF treatment, (1): IL-1β+ BLF+antimiR- NC and (2): IL-1β+ BLF+anti-miR-127-5p

Group pg·ml-1
IL-6 IL-10
IL-1β+BLF+anti-miR-NC 80.53±7.44 377.59±30.59
IL-1β+BLF+anti-miR-127-5p 207.82±22.31* 159.88±13.62*
t 16.237 19.505
p 0.000 0.000

Table 6: Effects of BLF and ANTI-miR-127-5p on IL-1β Induced Inflammatory Factors in Chondrocytes (x̄±s, n=9)

Substantial laboratory work has revealed that chondrocyte apoptosis and oxidative stress are closely associated with OA progression and development. Furthermore, chondrocyte inflammatory response might induce apoptosis and thus causes chondrocyte damage. IL-1β, a wellknown inflammatory cytokine, might contribute to the inflammatory response of chondrocytes, thereby mediating chondrocyte apoptosis[17]. Moreover, numerous miRNAs might participate in the process of OA development via modulating target gene expression[18]. Nevertheless, whether miRNAs can be used as a target for OA treatment with herbal medicines remains unknown.

With anti-inflammatory and anti-oxidative stress effects, BLF might attenuate rat inflammation model and hepatic ischemia-reperfusion injury[19,20]. But the influence of BLF on IL-1β-induced chondrocyte damage is not yet known. Herein, our data displayed that IL-1β might apparently improve chondrocyte apoptosis and cleaved caspase-3 and cleaved caspase-9 protein levels, consistent with the former report[21]. Furthermore, we found that BLF might relieve IL-1β-induced chondrocyte apoptosis and cleaved caspase-3 and cleaved caspase-9 in concentration-dependent manners, implying the repression of BLF on IL- 1β triggered chondrocyte apoptosis. In agreement with previous studies[22,23], our work presented that IL-1β treatment might decrease SOD activity and IL-10 content, and enhance LDH activity and IL-6 level. However, these influences were partially overturned by BLF exposure in a dose-dependent ways. These findings implied that BLF treatment might effectively weaken chondrocyte injury evoked by IL-1β.

miR-127-5p overexpression was demonstrated to abate OA progression via repressing chondrocyte apoptosis and inflammation[24,25]. Herein, BLF treatment might gradually ameliorate IL-1β mediated miR-127-5p content inhibition in chondrocytes, indicating BLF/miR-127-5p take part in regulating IL-1β mediated chondrocytes. Interfering miR-127-5p might partly recede the repression of BLF on IL-1β caused chondrocyte apoptosis, oxidative stress, and inflammation. The above observation validated that BLF treatment might constrain IL-1β evoked chondrocyte damage via upregulating miR-127-5p.

In summary, BLF might partly relieve IL-1β-induced chondrocyte apoptosis, oxidative stress, and inflammation through targeting miR-127- 5p, providing a new direction for OA treatment. However, the specific mechanism of action needs to be further explored.

Conflict of interests:

The authors declared no conflict of interests.

References