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. 2019 Jan 22;18(3):346–358. doi: 10.1080/15384101.2019.1568748

Diosgenin exerts its tumor suppressive function via inhibition of Cdc20 in osteosarcoma cells

Cheng Long a,✉, Juan Chen b, Hua Zhou c, Tao Jiang d, Xiang Fang a, Dong Hou e, Ping Liu e, Hong Duan a
PMCID: PMC6380388  PMID: 30640578

ABSTRACT

Osteosarcoma (OS) is one of the aggressive malignancies for young adults. Cdc20 (cell division cycle 20 homologue) has been reported to exhibit an oncogenic role in OS, suggesting that inhibition of Cdc20 could be a novel strategy for the treatment of OS. Since Cdc20 inhibitors have side effects, it is important to discover the new CDC20 inhibitors with non-toxic nature. In the present study, we determine whether natural agent diosgenin is an inhibitor of Cdc20 in OS cells. We performed MTT, FACS, Wound healing assay, Transwell, Western blotting, transfection assays in our study. We found diosgenin inhibited cell growth and induced apoptosis. Moreover, diosgenin exposure led to inhibition of cell migration and invasion. Notably, diosgenin inhibited the expression of Cdc20 in OS cells. Overexpression of Cdc20 abrogated the inhibition of cell growth and invasion induced by diosgenin. Our data reveal that inhibition of Cdc20 by diosgenin could be helpful for the treatment of patients with OS.

KEYWORDS: Diosgenin, osteosarcoma, Cdc20, cell growth, apoptosis

Introduction

Osteosarcoma (OS) is the common primary malignant bone tumor, which mainly affects children and adolescents [1]. The treatments of OS include surgical removal of cancerous lesion, chemotherapy such as cisplatin, doxorubicin, ifosfamide and methotrexate [2]. OS often has early systemic metastases, leading to poor prognosis [3]. The 5-year survival rate of OS patients with localized, non-metastatic disease is 60–70% [4]. However, OS patients with metastases have only 20% of 5-year survival rate [5]. The poor prognoses could be due to resistance to chemotherapeutic drugs [6]. To improve the treatment benefit of OS patients, it is required to discover the new therapeutic agents to treat OS.

Numerous studies have demonstrated that Cdc20 (cell division cycle 20) functions as an oncoprotein in the development and progression of human cancers [7]. Upregulation of Cdc20 was identified in various types of human cancers and was associated with poor prognosis [8–11]. For example, higher expression of Cdc20 was observed in glioblastomas patients, but not low-grade glioma patients [12]. Overexpression of Cdc20 is correlated to development and progression of hepatocellular carcinoma [13]. Moreover, Cdc20 was overexpressed in squamous cell carcinomas of the uterine cervix [8]. Notably, breast cancer patients with Cdc20 overexpression have short-team survival [14]. Similarly, Cdc20 overexpression is correlated with poor prognosis in oral squamous cell carcinoma [9], gastric cancer [15], urothelial bladder cancer [16], colorectal cancer [10], non-small cell lung cancer [17], and pancreatic cancer [18]. Therefore, targeting Cdc20 could be a promising way for treating human cancers.

Diosgenin, a steroid saponin of trigonella foenum graecum, has been reported to exert its antitumor activity in human cancer cells [19–21]. Diosgenin exhibits its anti-proliferative effect on different human cancer cells via activation of p53 and modulation of caspase-3 activity [22]. Diosgenin regulates the Akt, mTOR and JNK phosphorylation and suppresses fatty acid synthase in breast cancer cells [23,24]. In addition, diosgenin was found to inhibit the expression of cyclooxygenase-2 and 5-lipoxygenase pathways in colon cancer cells [25]. Moreover, diosgenin enhanced TRAIL-mediated apoptosis via activation of death receptor-5 in colon cancer cells [26]. Diosgenin inhibited Mdm2 and vimentin expression and led to suppression of HGF (hepatocyte growth factor)–induced EMT (epithelial-mesenchymal transition) in prostate cancer cells [27]. Similarly, diosgenin was observed to suppress migration and invasion via inhibition of matrix metalloproteinases expression in prostate cancer cells [28]. Diosgenin enhances the generation of ROS (reactive oxygen species) and modulation of mitochondrial pathway, leading to induction of apoptosis in liver cancer cells [29].

Several studies have demonstrated that diosgenin possesses tumor suppressive function in osteosarcoma cells [30–32]. For example, diosgenin treatment led to cell apoptosis, cell cycle arrest, and cyclooxygenases activity in OS cells [32]. Moreover, diosgenin exposure inhibited cell growth and induced apoptosis via activation of p53 in OS cells [31,33]. Although these studies have validated the function of diosgenin in OS, the molecular mechanism of diosgenin-mediated anti-proliferation of OS cells is unclear. Therefore, in the current study, we explored whether diosgenin could regulate the cell migration and invasion in OS cells. Due to that Cdc20 is an important oncogenic molecule in OS progression, we also determined whether diosgenin could inhibit the expression of Cdc20 in OS cells. Further, we dissected whether diosgenin exerts its anti-cancer activity via regulation of Cdc20 pathway. We found that diosgenin inhibited cell growth, induced apoptosis, suppressed cell migration and invasion in OS cells. We also found that diosgenin inhibited the expression of Cdc20, leading to anti-cancer activity in OS cells. Our results indicate that diosgenin could be an inhibitor of Cdc20 for the treatment of OS cells.

Results

Diosgenin inhibits OS cell growth

We measured the cell growth by MTT assay in OS cells after serial concentrations of diosgenin exposure for 48 h and 72 h. Our MTT results showed that diosgenin inhibited cell growth in a dose-dependent manner (Figure 1(A)). After 72 h treatments, 80 μM and 120 μM diosgenin exposures led to about 50% and 60% cell growth inhibition in U2OS cells, respectively (Figure 1(A)). Similarly, 80 μM and 120 μM diosgenin treatments caused about 40% and 50% cell growth suppression in MG63 cells, respectively (Figure 1(A)). Therefore, we will use 80 μM and 120 μM diosgenin treatments for our following study.

Figure 1.

Figure 1.

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Diosgenin exposure inhibits cell proliferation and induces apoptosis. (A) MTT assay was used to measure cell proliferation in OS cells after treatment with diosgenin for 48 h and 72 h, respectively. *P < 0.05, vs the control groups. (B) Cell apoptosis was evaluated by Flow cytometry in OS cells after diosgenin exposure for 48 h.

Diosgenin induces cell apoptosis

We further measured the cell apoptosis in OS cells after treatment with 80 μM and 120 μM for 48 h. We found that diosgenin treatment led to increased cell apoptosis in a dose-dependent manner in both U2OS and MG63 cell lines (Figure 1(B)). After 48 h treatments, 80 μM and 120 μM diosgenin exposures led to cell apoptosis from 2.94% in control group to 8.83% and 17.55% in U2OS cells, respectively (Figure 1(B)). Similarly, 80 μM and 120 μM diosgenin exposures increased cell apoptosis from 4.95% in control group to 11.71% and 31.95% cell in MG63 cells, respectively (Figure 1(B)). This finding suggests that diosgenin induced cell apoptosis in OS cells.

Diosgenin inhibits cell migration and invasion

Wound healing assay was used to determine whether diosgenin could suppress OS cell migration. We found that diosgenin inhibited cell migration in a dose-dependent manner in both OS cell lines (Figure 2(A,B)). Transwell invasion assay was used to further define whether diosgenin could retard cell invasion ability. We observed that the invaded cells, which were invaded through the pores of matrigel-coated membrane, were decreased in diosgenin-treated OS cells in a dose-dependent manner (Figure 2(C)). Taken together, diosgenin retarded cell migration and invasive properties in OS cells.

Figure 2.

Figure 2.

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Diosgenin inhibits migration and invasion in OS cells. (A) Wound healing assay was used to detect the cell migration in diosgenin-exposed OS cells. (B) Quantitative results are illustrated for panel A. *P < 0.05 and **P < 0.01, vs control. (C) Cell invasion was detected by Transwell chambers assay in diosgenin-exposed OS cells.

Diosgenin inhibits Cdc20 expression

Cdc20 has been known to play an oncogenic role in a variety of human cancers. Here, we determined whether diosgenin could inhibit the expression of Cdc20 in OS cells. Our real-time RT-PCR data demonstrated that diosgenin decreased the mRNA level of Cdc20 in OS cells (Figure 3(A)). The results from our western blotting analysis showed that diosgenin exposure led to inhibition of Cdc20 in a dose-dependent manner in OS cells (Figure 3(B,C)). These findings suggest that diosgenin inhibited the Cdc20 expression at the both transcription and translation levels. The expression of two downstream targets, p21 and Bim, was also measured in OS cells after diosgenin treatments for 72 h. We found that the levels of p21 and Bim were upregulated in diosgenin-treated OS cells (Figure 3(B,C)). This observation indicated that diosgenin could inhibit the expression of Cdc20 in OS cells.

Figure 3.

Figure 3.

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Diosgenin inhibited Cdc20 expression. (A) The mRNA level of Cdc20 was determined by real-time RT-PCR in OS cells after diosgenin treatment for 72 h. *P < 0.05, vs control. (B) Western blotting analysis was used to measure the expression of Cdc20, p21, and Bim in OS cells after diosgenin exposure for 72 h. (C) Quantitative results are illustrated for panel B. *P < 0.05 and **P < 0.01, vs control.

Over-expression of Cdc20 abolishes diosgenin-triggered cell growth inhibition and apoptosis

To further determine whether Cdc20 is involved in diosgenin-mediated cell growth suppression, Cdc20 cDNA constructor was transfected into OS cells in combination with diosgenin treatment. OS cells were tansfected with pcDNA 3.1 as control group. We found that overexpression of Cdc20 promoted cell growth in U2OS and MG63 cells (Figure 4(A)). Importantly, overexpression of Cdc20 abolished diosgenin-induced cell growth inhibition in both OS cell lines (Figure 4(A)). Next, cell apoptosis was measured in OS cells after diosgenin treatment plus Cdc20 cDNA constructor transfection. We observed that Cdc20 overexpression inhibited cell apoptosis in OS cells (Figure 4(B)). Notably, diosgenin-induced cell apoptosis was reversed to some extent after Cdc20 overexpression (Figure 4(B)). Our observations demonstrated that diosgenin exposure led to cell growth inhibition and apoptosis partly via down-regulation of Cdc20 in OS cells.

Figure 4.

Figure 4.

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Overexpression of Cdc20 abrogates diosgenin-mediated cell growth inhibition and apoptosis. (A) MTT assay was carried out to measure cell growth in OS cells after diosgenin treatment plus Cdc20 cDNA transfection. (B) Apoptotic cell death was accessed by Flow cytometry in OS cells after diosgenin treatment plus Cdc20 cDNA transfection. cDNA: Cdc20 cDNA transfection; Both: Cdc20 cDNA+Diosgenin. *P < 0.05, compared with control; # P < 0.05 compared with diosgenin treatment or Cdc20 cDNA transfection.

Over-expression of Cdc20 abrogates diosgenin-mediated inhibition of cell motility

In order to further dissect whether Cdc20 is involved in diosgenin-mediated suppression of migration, would healing assay was used to measure the migration in OS cells after diosgenin treatment in combination with Cdc20 cDNA constructor transfection. We found that overexpression of Cdc20 enhanced cell migration in OS cells and abrogated diosgenin-mediated reduction of cell migration (Figure 5(A,B)). Next, Transwell assay was used to analyze the cell invasion in OS cells after diosgenin exposure plus Cdc20 overexpression. Our results showed that overexpression of Cdc20 promoted cell invasion in OS cells (Figure 5(C,D)). Strikingly, Cdc20 overexpression abolished diosgenin-mediated inhibition of cell invasion in OS cells (Figure 5(C,D)). Overexpression of Cdc20 abolishes diosgenin-mediated inhibition of p21 and Bim.

Figure 5.

Figure 5.

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Overexpression of Cdc20 abolishes diosgenin-mediated inhibition of migration and invasion. (A) Cells migration was detected by wound healing assay in OS cells after diosgenin exposure and Cdc20 cDNA transfection. (B) Quantitative results are illustrated for the panel A. (C) Cell invasion was detected by Transwell chambers assay in OS cells after diosgenin treatment and Cdc20 cDNA transfection. (D) Quantitative results are illustrated for the panel C. cDNA: Cdc20 cDNA transfection; Both: Cdc20 cDNA+Diosgenin. *P < 0.05, compared with control; # P < 0.05 compared with diosgenin treatment or Cdc20 cDNA transfection.

We further measured the expression of Cdc20 and its downstream targets, p21 and Bim, in OS cells after Cdc20 cDNA transfection and diosgenin exposure. We found that Cdc20 cDNA constructor transfection increased the expression of Cdc20 in OS cells (Figure 6(A,B)). Our western blotting results also showed that overexpression of Cdc20 abrogated diosgenin-mediated suppression of Cdc20 in OS cells (Figure 6(A,B)). Moreover, Cdc20 overexpression reduced the level of p21 and Bim in OS cells (Figure 6(A,B)). Furthermore, Cdc20 cDNA transfection led to reversal of diosgenin-mediated upregulation of p21 and Bim in OS cells (Figure 6(A,B)). Our results imply that Cdc20 is associated with diosgenin-mediated anti-tumor activity.

Figure 6.

Figure 6.

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Overexpression of Cdc20 abolishes diosgenin-mediated upregulaton of p21 and Bim. (A) The expression of Cdc20, p21, and Bim in OS cells after diosgenin exposure and Cdc20 cDNA transfection was measured by western blotting analysis. (B) Quantitative results are illustrated for the panel A. Both: Cdc20 cDNA+Diosgenin. *P < 0.05, compared with control; # P < 0.05 compared with diosgenin treatment or Cdc20 cDNA transfection.

Downregulation of Cdc20 sensitizes OS cells to diosgenin treatment

To further explore whether Cdc20 plays a key role in diosgenin-mediated anti-cancer activity, OS cells were transfected with Cdc20 siRNA in presence of diosgenin exposure. Our MTT result showed that Cdc20 siRNA transfection inhibited cell growth in both OS cell lines (Figure 7(A)). Moreover, Cdc20 siRNA transfection in combination of diosgenin treatment led to cell growth inhibition to a greater degree in OS cells compared with diosgenin exposure alone or siRNA transfection alone (Figure 7(A)). In addition, Cdc20 siRNA transfection induced apoptosis from 5.24% in control group to 11.76% in U2OS cells, from 2.84% in control group to 17.87% in MG63 cells (Figure 7(B)). Altogether, downregulation of Cdc20 enhanced cell growth inhibition and apoptosis that were induced by diosgenin treatment.

Figure 7.

Figure 7.

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Downregulation of Cdc20 enhances diosgenin-mediated cell growth inhibition and apoptosis. (A) MTT assay was conducted to measure cell growth in OS cells after diosgenin treatment plus Cdc20 siRNA transfection. (B) Apoptotic cell death was accessed by Flow cytometry in OS cells after diosgenin treatment plus Cdc20 siRNA transfection. siRNA: Cdc20 siRNA transfection; Both: Cdc20 siRNA+Diosgenin. *P < 0.05, compared with control; # P < 0.05 compared with diosgenin treatment or Cdc20 siRNA transfection.

Down-regulation of Cdc20 enhances diosgenin-mediated suppression of cell motility

Our wound healing assay results demonstrated that down-regulation of Cdc20 inhibited cell migration in OS cells (Figure 8(A,B)). Moreover, down-regulation of Cdc20 enhanced diosgenin-induced inhibition of cell migration in both OS cell lines (Figure 8(A,B)). In line with this, down-regulation of Cdc20 retarded cell invasion in OS cell lines (Figure 8(C,D)). Consistently, Cdc20 siRNA transfection in combination with diosgenin triggered greater cell invasion inhibition than siRNA transfection alone or diosgenin treatment alone (Figure 8(C,D)). Our western blotting analysis result showed that Cdc20 siRNA transfection led to down-regulation of Cdc20 in OS cells (Figure 9(A,B)). Moreover, Cdc20 downregulation by its siRNA transfection increased the expression of p21 and Bim in OS cells (Figure 9(A,B)). Further, Cdc20 siRNA transfection enhanced upregulation of p21 and Bim induced by diosgenin exposures in OS cells (Figure 9(A,B)). Taken together, diosgenin induced tumor suppressive function in part via down-regulation of Cdc20 in OS cells.

Figure 8.

Figure 8.

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Downregulation of Cdc20 promotes diosgenin-mediated inhibition of migration and invasion. (A) Cells migration was detected by wound healing assay in OS cells after diosgenin exposure and Cdc20 siRNA transfection. (B) Quantitative results are illustrated for the panel A. (C) Cell invasion was detected by Transwell chambers assay in OS cells after diosgenin treatment and Cdc20 siRNA transfection. (D) Quantitative results are illustrated for the panel C. Both: Cdc20 siRNA+Diosgenin. *P < 0.05, compared with control; # P < 0.05 compared with diosgenin treatment or Cdc20 siRNA transfection.

Figure 9.

Figure 9.

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Downregulation of Cdc20 enhances diosgenin-mediated upregulaton of p21 and Bim. (A) The expression of Cdc20, p21, and Bim in OS cells after diosgenin exposure and Cdc20 siRNA transfection was measured by western blotting analysis. (B) Quantitative results are illustrated for the panel A. Both: Cdc20 siRNA+Diosgenin. *P < 0.05, compared with control; # P < 0.05 compared with diosgenin treatment or Cdc20 siRNA transfection.

Discussion

Accumulated evidence has demonstrated that diosgenin exhibits anti-cancer activity in various types of human cancers [21]. Diosgenin inhibited breast cancer stem-like cells via attenuation of the Wnt/beta-catenin signaling [34]. Diosgenin was found to inhibit mTOR signaling pathway and induce ROS-dependent autophagy in chronic myeloid leukemia cells [35]. Moreover, diosgenin exposure led to G2/M cell cycle arrest and apoptosis via inhibition of Akt phosphorylation and increased p21 and p27 expression in hepatocellular carcinoma cells [36]. Diosgenin also inhibited the hTERT (human telomerase reverse transcriptase) gene expression in lung cancer cells [37]. In line with these findings, our study showed that diosgenin inhibited cell growth and induced apoptosis in OS cells. Diosgenin inhibited cell migration via Vav2 phosphorylation and cdc42 activation in breast cancer cells [38]. Recently, diosgenin inhibited the expression of TAZ and suppressed tumor growth and invasion in hepatocellular carcinoma cells [39]. Similar findings were reported showing anti-invasive function of diosgenin in human hepatocellular carcinoma cells, gastric cancer, and prostate cancer [28,39,40]. Here, we also observed that diosgenin retarded cell migration and invasion in OS cells. Our study validates the anti-tumor activity of diosgenin in human OS cells.

Cdc20 has been known to exert its oncogenic function via targeting its some identified substrates, including Securin, Cyclin B1, Cyclin A, Nek2A, p21, Mcl-1 and Bim [7]. It has been reported that Cdc20 overexpression promoted cell growth and invasion and inhibited cell apoptosis in OS cells, suggesting that Cdc20 plays an oncogenic role in OS [41]. Therefore, targeting Cdc20 by its inhibitors might be a novel way to treat OS patients. TAME (tosyl-L-arginine methyl ester) decreased Cdc20 association with the APC, resulting in suppression of APC/C activity [42]. Moreover, pro-TAME has enhanced cell permeable activity and disrupts the APC-Cdc20/Cdh1 interaction, leading to inhibition of APC activation [42]. Notably, the APC/C inhibitor apcin, a small l cell-permeable molecule, blocks the interaction between APC/C and Cdc20 or Cdh1 [43], indicating that apcin prevents substrate from binding withCdc20. In fact, apcin has been shown to induce apoptotic cell death in multiple myeloma [44]. In addition, apcin might be an effective therapeutic agent targeting cohesion defective cancers [45]. Consistently, Cdc20 inhibitor apcin inhibited the growth and invasion of OS cells [46].

Recently, several natural compounds have been validated as Cdc20 inhibitors. For instance, ganodermanontriol inhibited the expression of Cdc20 and subsequently suppressed cell growth and invasiveness in breast cancer cells [47]. Compound 331 suppressed Cdc20 expression in glioma cells and caused cell death [48]. Withaferin A disrupted Mad2-Cdc20 complex in colorectal cancer cells [49]. One study showed that rottlerin decreased the expression of Cdc20 in glioma cells and led to inhibition of cell growth and invasion [50]. Another study defines that curcumin inhibited Cdc20 expression and led to suppression of cell survival in pancreatic cancer cells [51]. Our current study reveals that diosgenin reduced the expression of Cdc20, suggesting that diosgenin could be a promising agent for inhibition of Cdc20 for treating human OS patients with Cdc20 overexpression.

Materials and methods

Reagents

MTT [3-(4,5-dimethythi-azol- 2-yl)-2,5-diphenyl tetrazolium bromide], diosgenin, and Calcein-AM were purchased from Sigma-Aldrich (St Louis, MO, USA). Matrigel and Transwell inserts were obtained from BD Biosciences (Bedford, MA, USA). Annexin V-FITC/PI apoptosis detection kit was bought from Beyotime Biotechnology (Shanghai, China). Anti-Cdc20, anti-p21, anti-Bim, and anti-tubulin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). All secondary antibodies and lipofectamine 2000 were bought from Thermofisher Scientific (Waltham, MA, USA).

Cell culture

Human osteosarcoma cell lines U2OS and MG63 were cultured in DMEM (Thermo Fisher Scientific, USA), supplemented with 10% FBS (fetal bovine serum, HyClone, USA) and penicillin and streptomycin (100 U/ml, Thermo Fisher Scientific, USA), and maintained in a 5% CO2 atmosphere at 37°C.

MTT assay for cell viability

OS cells were seeded in 96-well plates for overnight incubation. Cells were then treated with different concentrations of diosgenin for 48 h and 72 h, respectively. In control group, cells were exposed to 0.1% DMSO. Then, 10 μL of MTT (5 mg/ml) reagent was added and incubated for 4 h at 37°C. The supernatant was removed and 100 μl DMSO was added for 15 min at room temperature. The absorption of each well was measured by a microplate reader at 490 nm.

Flow cytometer analysis for cell apoptosis

OS cells were seeded in six-well plates for overnight incubation. After cells were treated with 80 μM and 120 μM of diosgenin for 48 h, respectively, cells were collected and re-suspended in binding buffer with propidium iodide (PI) and FITC-conjugated anti-Annexin V antibodies for 15 min at room temperature under dark conditions.The FACS calibur flow cytometer (BD, USA) was used to measure the cell apoptosis.

Wound healing assay

OS cells were seeded in 6-well plates for incubation until cells reached to more than 90% confluence. The scratch wounds were created using a yellow pipette tip on the surface cells. Then, cells were treated with diosgenin for 20 hours and the width of the wound images was taken at 0 h and 20 h, respectively.

Cell invasion assay

Diosgenin-treated OS cells were incubated in serum-free DMEM in the upper-chamber of the Transwell inserts that was precoated with Matrigel. Meanwhile, complete DMEM (10% FBS) was added in the lower-chamber. After 20 h incubation, the invaded cells through the chamber member pores were stained to Calcein-AM for 30 min. Then, a fluorescence microscope was used to take the stained OS cells.

Transfection of plasmids and small interfering RNAs (siRNAs)

To further define the role of Cdc20 in diosgenin-mediated anti-tumor function, Cdc20 expression vector pcDNA3.1-Cdc20 or Cdc20 siRNA was transfected into OS cells by Lipofectamine® 2000 reagent following the manufacturer’s instructions.

Real-time reverse transcription-PCR (RT-PCR) analysis

The total RNA was extracted by Trizol reagent. RT-PCR was conducted using SYBR Green PCR Master Mix as described before [52]. The primers used in PCR reaction are: Cdc20, forward primer 5′-GAC CAC TCC TAG CAA ACC TGG-3′ and reverse primer 5′-GGG CGT CTG GCT GTT TTC A-3′; GAPDH, forward primer 5′-ACC CAG AAG ACT GTG GAT GG-3′; reverse primer 5′- CAG TGA GCT TCC CGT TCA G- 3′.

Western blotting assay

Cells were harvested after diosgenin treatment or Cdc20 siRNA transfection or Cdc20 cDNA constructor transfection and re-suspended in protein lysis buffer. Protein concentrations were detected by the BCA Protein Assay. Then 30 μg of protein was separated by SDS-PAGE and transferred to PVDF membrane. The membrane was blocked by 5% nonfat milk for 1 h, and incubated with primary antibody at 4°C for overnight. The membrane was washed and incubated with secondary antibody for 1 h at room temperature. ECL assay was used to measure the protein level.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 5.0 Software (La Jolla, CA, USA). Two-tailed Student’s t-test was used to analyze statistical significance between two groups. ANOVA was used to measure difference between multiple groups. All data were presented with means ± standard deviation (S.D.). P < 0.05 was considered to be statistically significant.

Acknowledgments

This work was support by Key research and development projects of Sichuan science and technology department grant 2017SZ0127.

Disclosure statement

No potential conflict of interest was reported by the authors.

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