RNA-sequencing analysis reveals the potential mechanism by which a Dioscorea polystachya (Chinese yam) protein ameliorates hyperglycemia-induced endothelial dysfunction

doi:10.21203/rs.3.rs-3315991/v1

Download PDF

Research Article

RNA-sequencing analysis reveals the potential mechanism by which a Dioscorea polystachya (Chinese yam) protein ameliorates hyperglycemia-induced endothelial dysfunction

https://doi.org/10.21203/rs.3.rs-3315991/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Hyperglycemia-induced endothelial dysfunction is one of the most crucial factors leading to disability and death in patients with diabetes mellitus (DM). Dioscorea polystachya (Chinese yam), a well-known ingredient used in traditional Chinese medicine, contains various bioactive components that have been widely used to treat endothelium-related diseases. The aim of this study was to explore the molecular mechanism and therapeutic targets of the Chinese yam protein DP1 on hyperglycemia-induced endothelial dysfunction.

Methods and results

We first investigated the effect of DP1 isolated from Chinese yam, on human umbilical vein endothelial cells (HUVECs) that had been exposed to high-glucose (HG, 30 mmol/L) conditions for 48 h. CCK-8 assay showed that DP1 enhanced HUVECs viability. ELISA and tube formation assays indicated that DP1 increased NO levels and promoted angiogenesis by increasing node numbers, mesh numbers, and tube length. Moreover, SA-β-gal staining showed that DP1 inhibited the HG-induced increase in the percentage of senescent cells. RNA-sequencing analysis revealed a total of 335 DEGs between the HG group and the DP1 group. Gene expression profiling showed that NUMBL, AFDN, ITGA6, ITGB1, DSP, AGO2, QKI, FBXW7, YAP1, CCBE1, NCL, SIRT1, JUND, PRKDC, PTEN, CDK6, KAT6A, NFATC3, PPP1CA, and MIF might play key roles in mediating the effects of DP1 treatment on HG-induced endothelial dysfunction by regulating angiogenesis and cellular senescence, and these findings were validated by RT-PCR.

Conclusion

This study deepens our current knowledge of the molecular effects of DP1 on hyperglycemia-induced endothelial dysfunction and suggests a possible strategy for treating endothelium-related diseases using a Chinese yam protein.

Chinese yam protein

diabetes mellitus

endothelial dysfunction

angiogenesis

cellular senescence

Diabetes mellitus (DM) is a serious condition that impacts blood vessels, leading to a two- to fourfold rise in the incidence of vascular complications compared with healthy individuals, and can be fatal [1]. In 2050, there will be 1.31 billion people living with DM worldwide [2]. Hyperglycemia-induced endothelial dysfunction is a precursor and initiating factor of vascular complications in patients with DM [3]. The endothelium, a monolayer of cells situated on the inner surface of blood vessels, plays a pivotal role in preserving vascular homeostasis by promoting angiogenesis and generating active substances, such as nitric oxide (NO), that regulating the balance between vasoconstriction and vasodilation [4]. Once hyperglycemia impairs endothelial function, a cascade of microvascular and macrovascular disorders, including but not limited to myocardial infarction, atherosclerosis, and erectile dysfunction, is triggered by compromised vascular barrier structure and aberrant vascular tone [5]. Consequently, it is imperative to prevent and treat hyperglycemia-induced endothelial dysfunction.

While the pathogenesis of hyperglycemia-induced endothelial dysfunction is multifactorial, cellular senescence is a noteworthy event in the development of endothelial dysfunction in individuals with DM [6–7]. Prolonged exposure to hyperglycemic conditions may impede the endothelium's capacity for growth and proliferation, resulting in expedited cellular senescence, which reduces angiogenesis, induces endothelial dysfunction, and weakens vascular barrier function [8]. Studies in humans and animals have shown that endothelial senescence occurs in diabetic-induced atherosclerosis, and inhibition of hyperglycemia-induced endothelial senescence can attenuate endothelial dysfunction and atherosclerosis [9–10]. Therefore, protecting endothelial cells from senescence is clearly an effective approach for alleviating hyperglycemia-induced endothelial dysfunction.

Dioscorea polystachya (Chinese yam) is a well-known edible tuber crop and ingredient used in traditional Chinese medicine that has been extensively studied and applied in the treatment of endothelium-related diseases, such as diabetes and atherosclerosis, due to being rich in nutritional and bioactive components [11–14]. DP1, a protein that can be extracted, isolated, and purified from Chinese yam, has been shown to improve many conditions, such as testicular dysfunction and erectile dysfunction [15–16]. Recently, DP1 has been reported to alleviate the injury to cavernous endothelial cells, a type of microvascular endothelial cell, caused by high-glucose (HG) [17]. Therefore, we hypothesized that DP1 would also protect endothelial cells in large vessels, such as human umbilical vein endothelial cells (HUVECs), from hyperglycemia-induced injury.

In this study, we investigated the effect of DP1 on HG-induced HUVEC injury and performed RNA-sequencing to identify the molecular basis of this effect and elucidate the potential mechanism by which DP1 protects against endothelial dysfunction.

DP1 preparation

Chinese yam (Dioscorea polystachya) was purchased from Jiaozuo, Henan Province, and identified by Professor Zhe Wang of Changchun University of Chinese Medicine (taxonomic serial number: 810115). DP1 was extracted, isolated, and purified from the Chinese yam, as previously described [15].

Cell culture

HUVECs were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in F-12K medium (Procell) containing 10% fetal bovine serum (Gibco Laboratories, NY, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, MO, USA) at 37°C in a humidified 5% CO₂ atmosphere.

Cell viability assay

A cell counting kit-8 (CCK-8, Boster, Wuhan, China) was used to detect HUVECs viability under different conditions. First, 3×10³ HUVECs were seeded in 96-well plates and incubated at 37°C with 5% CO₂ overnight. Then, the cells were starved in F-12K medium for 24 h. Next, the medium was replaced with HG (30 mmol/L D-glucose) with or without 5, 10, or 15 µg/mL DP1, and the cells were re-incubated at 37°C. After 48 h, 10 µL CCK-8 was added to each well, and the plates were incubated in darkness at 37°C for 1 h. Finally, the optical density was measured at 450 nm using a microplate reader (Infinite 200 pro, TECAN, Switzerland).

NO level determination

First, 3×10³ HUVECs were seeded in 6-well plates and incubated overnight at 37°C with 5% CO₂. The cells were then cultured in F-12K medium for 24 h to create starvation conditions. Next, the medium was replaced with 30 mmol/L HG with or without 15 µg/mL DP1, and the cells were re-incubated at 37°C for 48 h. Finally, the conditioned medium was collected, and the NO content was determined using an enzyme-linked immunosorbent assay (ELISA) kit (Sinobestbio, Shanghai, China) according to the manufacturer’s protocol.

Tube formation assay

Fifty microliters of Matrigel (Corning, NY, USA) was added to 96-cell plates that had been pre-cooled to 4°C, and the plates were then incubated at 37°C for 30 min. Next, HG-cultured HUVECs with or without 15 µg/mL DP1 were added to the Matrigel at 2×10⁴ cells per well and incubated for 12 h. At the end of the incubation period, the tube-like structures that had formed in each well were photographed using an EVOS® FL Auto Imaging System (Thermo Fisher Scientific, MA, USA) and analyzed with Image J software (National Institutes of Health, Bethesda, USA).

Senescence-associated β-galactosidase (SA-β-gal) staining

First, 3×10³ HUVECs were seeded in 6-well plates and incubated overnight at 37°C with 5% CO₂. The cells were then cultured in F-12K medium for 24 h to create starvation conditions. Next, the medium was replaced with 30 mmol/L HG with or without 15 µg/mL DP1, and the cells were re-incubated at 37°C for 48 h. Then, the cells in each group were washed once with PBS, fixed with 1 mL fixative at 37°C for 15 min, and then stained with 1 mL β-gal solution. Finally, the cells were incubated overnight at 37°C and photographed using a microscope. The images were analyzed using ImageJ software.

RNA extraction and sequencing

The cells in each group were washed three times with phosphate-buffered saline (PBS) and lysed with TRIzol reagent (Invitrogen, CA, USA), and total RNA was obtained. The RNA integrity and quality were assessed using an Agilent 2100 bioanalyzer and a Nano Drop (Thermo Fisher Scientific). mRNA was enriched from the total RNA using Oligo(dT) magnetic beads and fragmented into short pieces with fragmentation buffer. The mRNA fragments were then used to synthesize double-stranded cDNA, followed by PCR amplification and preparation of an RNA-seq library. RNA-seq was carried out on an Illumina HiSeq 2500 platform (Illumina, CA, USA).

RNA-seq data analysis

After the RNA-seq results had been obtained, high-quality clean reads were identified using Illumina HCS (v1.1) to remove adapter/poly-N sequences and low-quality reads. The clean reads in each sample were aligned to the human reference genome using HISAT (D2015), and gene expression levels were calculated using RSEM (v1.2.12). Genes with a fold change of ≥ 1.2 or ≤ − 1.2 and an adjusted P-value < 0.05, as determined using the DESeq2 R package (v1.16.1), were defined as differentially expressed genes (DEGs).

Function and pathway enrichment analysis

Gene ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs between the HG group and DP1 group were conducted using Cluster Profiler (an R package, v4.0.2). Results with a corrected P-value < 0.05 were considered to be significantly enriched in the DEGs.

RT-PCR validation of RNA-seq data

Total RNA was obtained from each group and synthesized into cDNA using a Prime Script RT Reagent Kit (DRR047A; Takara, Shiga, Japan). The expression levels of the DGEs were measured using quantitative RT-PCR using SYBR Green PCR Master Mix (DRR420A; Takara) and an iCycler-iQ Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, USA). DEG expression levels were calculated using the 2^−ΔΔCt method and normalized to the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH). The primer sequences are listed in Supplementary Table S1.

Statistical analysis

All experiments were carried out with three biological replicates, and the results are expressed as the mean ± S.D. All data were analyzed by one-way ANOVA, followed by the Tukey-Kramer multiple comparison test using GraphPad Prism software (v6.0). P < 0.05 was considered to be statistically significant.

DP1 enhanced cell viability and endothelial function in HUVECs exposed to HG conditions

A CCK-8 assay was performed to investigate the effects of DP1 on cell viability. HUVECs subjected to HG conditions exhibited a significant decrease in cell viability compared with the control group (Fig. 1A). Treatment with DP1 (5, 10, or 15 µg/mL) increased cell viability in a concentration-dependent manner. The optimal concentration of DP1 was 15 µg/mL.

We next detected the effects of DP1 on NO levels and tube formation in HUVECs exposed to HG conditions. Treatment with 15 µg/mL DP1 significantly increased NO levels in the HG-injured HUVECs and promoted tube formation, including an increase in node numbers, mesh numbers, and tube length (Fig. 1B-F).

DP1 prevented cell senescence in HUVECs exposed to HG conditions

We further studied the effect of DP1 on cell senescence in HG-injured HUVECs. SA-β-gal staining was used to label senescent cells. HG resulted conditions induced senescence in 79.26 ± 4.61% of the cells (Fig. 2). Treatment with 15 µg/mL DP1 inhibited HG-induced HUVEC senescence, reducing the percentage of senescent cells to 34.47 ± 3.60% (Fig. 2).

Statistical summary of the RNA-seq data

We performed Illumina sequencing to analyze the transcriptome of HG-treated HUVECs with or without DP1. The RNA-seq data for each group is briefly summarized in Table 2. In total, 477,774 and 468,538 clean reads were obtained from the HG group and the DP1 group, respectively, after filtering out low-quality reads. More than 94% and 97% of the reads met the Q30 and Q20 standards, respectively. The GC content was around 51–52% in each group. More than 96% of the clean reads from the HG and DP1-treated samples mapped to the human genome.

Table 2

Summary of RNA-seq sequence assembly results
	HG group	DP1 group
Clean read number	477,774	468,538
Q30 (%)	94.14	94.09
Q20 (%)	97.90	97.88
GC (%)	52.12	51.12
Total mapped reads (%)	97.04	96.61
Unique mapped reads (%)	89.00	89.16
Multiple mapped reads (%)	8.04	7.45
Note: Total mapped reads (%) = Unique mapped reads (%) + Multiple mapped reads (%).

Identification of DEGs

DEGs between the HG group and the DP1 group were identified and are shown in Table 3. A total of 335 DEGs were detected, including 212 genes that were upregulated and 123 genes that were downregulated in the DP1 group compared with the HG group. All the DEGs were listed in Supplementary Table S2.

Table 3

DEGs between HG group and DP1 group
Genes	Number
Total	335
Upregulated	212
Downregulated	123

Functional and pathway enrichment analysis

To understand the function of the genes that may be involved in DP1-mediated amelioration of HG-induced endothelial dysfunction, GO enrichment analysis of the DEGs was performed for. The top 10 GO terms with the highest enrichment factors related to biological processes, cellular components, and molecular functions are shown in Supplementary Fig. S1A. The enriched GO biological processes were mainly cell processes (GO: 0009987), metabolic processes (GO: 0008152), and biological regulation (GO: 0065007). The most highly enriched GO cellular components were organelles (GO: 0043226), membrane (GO: 0016020), and protein-containing complexes (GO: 0032991). The enriched GO molecular functions were mainly catalytic activity (GO: 1902494), structural molecule activity (GO: 0005198), and molecular function regulator (GO: 0098772). Next, we performed KEGG pathway enrichment analysis to identify interactions among and possible biological pathways associated with the DEGs. The top 30 enriched pathways are shown in Supplementary Fig. S1B. The enriched pathways were mainly the FoxO signaling pathway (ko04068), the cGMP-PKG signaling pathway (ko04022), and the cellular senescence pathway (ko04218).

DP1 increased the expression levels of DEGs involved in angiogenesis

To gain insight into the molecular mechanisms by which DP1 promotes HUVEC angiogenesis after HG injury, we further analyzed the expression levels of DEGs involved in regulating angiogenesis. As shown in Table 4, the expression levels of NUMB-like endocytic adaptor protein (NUMBL), afadin (AFDN), integrin subunit alpha 6 (ITGA6), integrin subunit beta 1 (ITGB1), desmoplakin (DSP), argonaute RISC catalytic component 2 (AGO2), KH domain–containing RNA-binding (QKI), F-box and WD repeat domain–containing 7 (FBXW7), Yes1-associated transcriptional regulator (YAP1), collagen- and calcium-binding EGF domains 1 (CCBE1), and nucleolin (NCL) were significantly increased by DP1 treatment.

Table 4

Identification of DEGs involved in angiogenesis
Gene ID	Gene name	Expression level (FPKM)		Log₂ fold change (DP1/HG)	P-value
Gene ID	Gene name	HG	DP1	Log₂ fold change (DP1/HG)	P-value
NUMBL	NUMB-like endocytic adaptor protein	64.71	220.05	0.45	1.29E-03
AFDN	afadin	742.62	1014.03	0.31	2.18E-02
ITGA6	integrin subunit alpha 6	2191.04	2851.79	0.30	9.76E-03
ITGB1	integrin subunit beta 1	4287.51	5888.53	0.37	5.26E-04
DSP	desmoplakin	1333.94	1875.75	0.37	2.56E-03
AGO2	argonaute RISC catalytic component 2	794.39	1089.35	0.32	1.79E-02
QKI	KH domain–containing RNA-binding	1021.36	1339.89	0.27	3.67E-02
FBXW7	F-box and WD repeat domain–containing 7	85.61	174.16	0.31	3.19E-02
YAP1	Yes1-associated transcriptional regulator	1564.89	2133.54	0.35	3.29E-03
CCBE1	collagen- and calcium-binding EGF domains 1	1430.5	1913.72	0.32	7.41E-03
NCL	nucleolin	7473.03	9363.31	0.27	4.43E-03

DP1 treatment alters the expression levels of DEGs involved in cellular senescence

Nine genes involved in cellular senescence were identified among the DEGs, including sirtuin 1 (SIRT1), AP-1 transcription factor subunit (JUND), protein kinase DNA-activated catalytic subunit (PRKDC), phosphatase and tensin homolog (PTEN), cyclin-dependent kinase 6 (CDK6), lysine acetyltransferase 6A (KAT6A), nuclear factor of activated T cells 3 (NFATC3), protein phosphatase 1 catalytic subunit alpha (PPP1CA), and macrophage migration inhibitory factor (MIF), as shown in Table 5.

Table 5

Identification of DEGs involved in cellular senescence
Gene ID	Gene name	Expression level (FPKM)		Log₂ fold change (DP1/HG)	P-value
Gene ID	Gene name	HG	DP1	Log₂ fold change (DP1/HG)	P-value
SIRT1	sirtuin 1	222.99	380.95	0.36	1.73E-02
JUND	AP-1 transcription factor subunit	586.33	825.69	0.32	2.43E-02
PRKDC	protein kinase, DNA-activated, catalytic subunit	2670.86	3790.96	0.41	1.13E-04
PTEN	phosphatase and tensin homolog	322.53	487.26	0.33	2.95E-02
CDK6	cyclin-dependent kinase 6	551.49	845.58	0.40	4.94E-03
KAT6A	lysine acetyltransferase 6A	531.58	731.49	0.30	3.96E-02
NFATC3	nuclear factor of activated T cells 3	403.17	580.6	0.31	3.75E-02
PPP1CA	protein phosphatase 1 catalytic subunit alpha	4566.24	3678.06	-0.29	6.90E-03
MIF	macrophage migration inhibitory factor	7723.89	6237.84	-0.29	4.17E-03

RT-PCR validation of RNA-seq data

The expression levels of 11 DEGs involved in angiogenesis and nine DEGs involved in cellular senescence were validated by RT-PCR (Fig. 3). The 2^−ΔΔCt method was used to calculate the relative gene expression levels by normalizing to GAPDH. As shown in Fig. 3, the expression levels of the identified DEGs as measured by RT-PCR were consistent with the results from the RNA-sequencing analysis.

Endothelial dysfunction is a key pathological mechanism underlying diabetes-induced vascular disease [18]. Therefore, the search for safe and effective drugs to treat hyperglycemia-induced endothelial dysfunction is currently of considerable interest in the field of diabetes and cardiovascular disease research. Chinese yam, an ingredient used in traditional Chinese medicine, is widely used in clinical practice to treat endothelium-related diseases. However, little is known about the mechanism by which it exerts its beneficial effects in hyperglycemia-induced endothelial dysfunction. In the present study, we found that DP1, a Chinese yam protein, has a remarkable ability to alleviate HG-induced HUVEC functional impairment and cellular senescence. We used RNA-seq analysis to identify crucial targets of DP1 that promote angiogenesis and inhibit cell senescence. Our findings provide new insight into the mechanisms underlying how Chinese yam ameliorates endothelial dysfunction.

The vascular endothelium is a multifunctional organ that maintains vascular integrity and regulates vascular homeostasis; these functions rely on the angiogenic capacity of vascular endothelial cells and their production of active substances such as NO [19]. Previous studies have indicated that the impaired endothelial function underlying both macro- and microvascular complications can be caused by prolonged, transient, and acute hyperglycemia in animal models as well as in humans [20]. In our study, HUVECs exposed to HG conditions exhibited decreased proliferation, lower NO levels, and impaired tube formation compared with uninjured control cells, making them an ideal in vitro model of diabetes-induced endothelial dysfunction [21]. Treatment with DP1 significantly improved HUVEC viability, NO production, and angiogenic capacity. These results suggest that DP1 can restore HG-induced functional damage to HUVECs.

Cellular senescence indicates a permanent arrest of cell growth and proliferation and is a major factor contributing to hyperglycemia-induced endothelial dysfunction owing to the disruption in endothelial cell permeability and motility, decreased NO production, and abnormal gene expression [22–24]. At the phenotypic level, upregulation of SA-β-gal activity is a key feature of senescent endothelial cells [25]. To investigate whether DP1 attenuates HG-induced endothelial dysfunction by inhibiting SA-β-gal activity, we performed SA-β-gal staining of HUVECs. Consistent with a previous study, we found that the number of SA-β-gal–positive cells was significantly increased under HG conditions compared with normal glucose conditions [26]. Treatment with DP1 decreased the percentage of senescent cells, indicating that the beneficial effects of DP1 on HG-induced endothelial dysfunction are associated with inhibition of cellular senescence.

To determine the molecular mechanism by which DP1 protects against HG-induced endothelial dysfunction, we performed RNA-seq. In total, 335 DEGs were identified between the HG group and the DP1 group, including 212 upregulated genes and 123 downregulated genes. From these DEGs, we selected 11 angiogenesis-related genes that were upregulated in the DP1 group as potential factors associated with HG-induced endothelial dysfunction. For instance, NUMBL, AFDN, ITGA6, ITGB1, and DSP encode cell adhesion–related factors that play crucial roles in angiogenesis by regulating the interaction between endothelial cells and extracellular matrix or other cells [27–31]. Silencing AGO2, which encodes a key participant in mRNA biogenesis, inhibits HUVECs angiogenesis and enhances HUVECs apoptosis [32]. QK1 is a component of the endothelial cell cycle progression post-transcriptional regulatory network and is crucial to angiogenesis [33]. FBXW7, a SCF protein complex component responsible for polyubiquitination, promotes angiogenesis by limiting endothelial Notch activity [34]. YAP1 is a transcriptional co-activator that regulates angiogenesis by controlling mitochondrial biogenesis [35]. CCBE1 promotes angiogenic sprouting via endothelial cell migration during embryogenesis [36–37]. NCL acts as a multifunctional DNA-, RNA-, and protein-binding protein in the nucleolus of eukaryotic cells, and binds to the proangiogenic factors VEGF-A and MMP-9 [38]. Our results showed that DP1 regulates the expression levels of genes involved in angiogenesis that can contribute to endothelial dysfunction in the context of hyperglycemia. These results indicated that DP1 may protect against HG-induced endothelial dysfunction by promoting angiogenesis.

In addition to the above findings, we also identified nine DEGs that involved in HG-induced endothelial cellular senescence, including SIRT1, JUND, PRKDC, PTEN, CDK6, KAT6A, NFATC3, PPP1CA, and MIF. SIRT1 is a member of the sirtuin family of NAD⁺-dependent deacylases and participates in HG-induced HUVEC senescence by regulating FoxO-1/p53 acetylation and p21 expression [39]. JUND is considered to be an important modulator of oxidative stress and plays a key role in regulating cell growth and survival during endothelial recovery [40]. PRKDC, also known as DNA-dependent protein kinase catalytic subunit (DNAPKcs), is a central regulator of DNA end access and can enhance cell proliferation [41–42]. PENT and CDK6 play crucial roles in cell proliferation and the cell cycle, and their absence induces HUVEC senescence [43–44]. KAT6A, a member of the monocytic leukemia zinc-finger protein family, is a potent inhibitor of cellular senescence that acts via the INK4A-ARF pathway [45]. NFATC3, nuclear factor of activated T cells, affects senescence by regulating Ca²⁺ signaling [46]. PPP1CA, the catalytic subunit of protein phosphatase 1a, regulates senescence in a pRb-dependent manner [47]. MIF is a proinflammatory cytokine that controls cellular senescence by regulating p53 and NF-κb signaling [48]. These results suggested that DP1 alleviates HG-induced endothelial dysfunction by regulating multiple cellular senescence-related factors.

In summary, this study demonstrated that DP1, a bioactive protein component of Chinese yams, can ameliorate HG-induced endothelial dysfunction by promoting angiogenesis and inhibiting cellular senescence. These results greatly expanded our knowledge of the molecular mechanism by which treatment with Chinese yam improves endothelial function. Our findings also suggest possible strategies for preventing and treating endothelial dysfunction, and related cardiovascular disease, using a protein from the Chinese yam.

Ethical approval

No applicable.

Conflict of interest

The authors declare that they have no conflict of interest.

Authors’ contributions

All authors contributed to the study conception and design. SL and ML conceived and designed this research. SY and XX performed the experiments. LJ, JF and HZ analyzed the data. SY, XX and SL wrote the first draft of manuscript. DZ and SW performed the review and modification of the manuscript. All authors reviewed and approved the final version of the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the Scientific and Technological Development Planning Foundation of Jilin Province (YDZJ202301ZYTS445), the National College Students' innovation and entrepreneurship training program (202210199016X) and the College Students' innovation and entrepreneurship training program of Jilin Province (S202210199011X).

Acknowledgment

We thank Emily Crow, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the English text of a draft of this manuscript.

Availability of data and materials

No applicable.

Shi Y, Vanhoutte PM (2017) Macro- and microvascular endothelial dysfunction in diabetes. J Diabetes 9: 434-449. https://doi:10.1111/1753-0407.12521.
GBD 2021 Diabetes Collaborators (2023). Global, regional, and national burden of diabetes from 1990 to 2021, with projections of prevalence to 2050: a systematic analysis for the Global Burden of Disease Study 2021. Lancet (London, England), 402(10397), 203–234. https://doi.org/10.1016/S0140-6736(23)01301-6.
Jia G, Sowers JR (2014) Endothelial dysfunction potentially interacts with impaired glucose metabolism to increase cardiovascular risk. Hypertension 64: 1192–1193. https://doi.org/10.1161/HYPERTENSIONAHA.114.04348.
Aird WC (2007) Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms. Circ Res 100: 158-173. https://doi:10.1161/01.RES.0000255691.76142.4a.
Ingelfinger JR, Jarcho JA (2017) Increase in the Incidence of Diabetes and Its Implications. N Engl J Med 376: 1473-1474. https://doi:10.1056/NEJMe1616575.
Khemais-Benkhiat S, Belcastro E, Idris-Khodja N, Park, SH, Amoura, L, Abbas, M, Auger, C, Kessler, L, Mayoux, E, Toti, F, Schini-Kerth, VB (2020) Angiotensin II-induced redox-sensitive SGLT1 and 2 expression promotes high glucose-induced endothelial cell senescence. J Cell Mol Med 24: 2109-2122. https://doi:10.1111/jcmm.14233.
Zhang T, Tian F, Wang J, Zhou S, Dong X, Guo K, Jing J, Zhou Y, Chen Y (2015) Donepezil attenuates high glucose-accelerated senescence in human umbilical vein endothelial cells through SIRT1 activation. Cell Stress Chaperones 20: 787-792. https://doi:10.1007/s12192-015-0601-4.
Chen T, Ma C, Fan G, Liu H, Lin X, Li J, Li N, Wang S, Zeng M, Zhang Y, Bu P (2021) SIRT3 protects endothelial cells from high glucose-induced senescence and dysfunction via the p53 pathway. Life Sci 264: 118724. https://doi.org/10.1016/j.lfs.2020.118724.
Stojanović SD, Fiedler J, Bauersachs J, Thum T, Sedding, DG (2020) Senescence-induced inflammation: an important player and key therapeutic target in atherosclerosis. Eur Heart J 41: 2983–2996. https://doi.org/10.1093/eurheartj/ehz919.
Wang G, Han B, Zhang R, Liu Q, Wang X, Huang X, Liu D, Qiao W, Yang M, Luo X, Hou J, Yu B (2021) C1q/TNF-Related Protein 9 Attenuates Atherosclerosis by Inhibiting Hyperglycemia-Induced Endothelial Cell Senescence Through the AMPKα/KLF4 Signaling Pathway. Front Pharmacol 12: 758792. https://doi.org/10.3389/fphar.2021.758792.
Epping J, Laibach N (2020) An underutilized orphan tuber crop-Chinese yam: a review. Planta 252: 58. https://doi.org/10.1007/s00425-020-03458-3.
Pang SQ, Luo ZT, Wang CC, Hong XP, Zhou J, Chen F, Ge L, Li X, Dai YL, Wu YL, Zhang JH (2020) Effects of Dioscorea polystachya 'yam gruel' on the cognitive function of diabetic rats with focal cerebral ischemia-reperfusion injury via the gut-brain axis. J Integr Neurosci 19: 273–283. https://doi.org/10.31083/j.jin.2020.02.69.
Lee W, Jeong SY, Gu MJ, Lim JS, Park EK, Baek MC, Kim JS, Hahn D, Bae JS (2019) Inhibitory effects of compounds isolated from Dioscorea batatas Decne peel on particulate matter-induced pulmonary injury in mice. J Toxicol Environ Health A 82: 727–740. https://doi.org/10.1080/15287394.2019.1646174.
Wu FC, Jiang JG (2019) Effects of diosgenin and its derivatives on atherosclerosis. Food Func 10： 7022–7036. https://doi.org/10.1039/c9fo00749k.
Yu S, Han B, Xing X, Li Y, Zhao D, Liu M, Wang S (2021) A Protein from Dioscorea polystachya (Chinese Yam) Improves Hydrocortisone-Induced Testicular Dysfunction by Alleviating Leydig Cell Injury via Upregulation of the Nrf2 Pathway. Oxid Med Cell Longev 2021: 3575016. https://doi.org/10.1155/2021/3575016.
Li Y, Zhao D, Wang S, Liu M, Yu S (2022) PI3K/Akt/eNOS pathway mediated the amelioration effect of Chinese yam protein on penile erectile dysfunction in kidney-yang deficiency rats. Chinese Journal of Hospital Pharmacy 42: 2607-2611. https://doi:10.13286/j.1001-5213.2022.24.06.
Xing X, Zhao D, Wang S, Yu S, Li Y, Liu M (2021) Based on RNA-Seq technology to analyze the mechanism of yam protein in prevention and treatment diabetes-induced erectile dysfunction. China Pharmacy 32: 2859-2868. https://doi:10.6039/j.issn.1001-0408.2021.23.08.
Pi X, Xie L, Patterson C (2018) Emerging Roles of Vascular Endothelium in Metabolic Homeostasis. Circulation research 123: 477–494. https://doi.org/10.1161/CIRCRESAHA.118.313237
Godo S, Shimokawa H (2017) Endothelial Functions. Arteriosclerosis, thrombosis, and vascular biology 37: e108–e114. https://doi.org/10.1161/ATVBAHA.117.309813.
Kaur R, Kaur M, Singh J (2018) Endothelial dysfunction and platelet hyperactivity in type 2 diabetes mellitus: molecular insights and therapeutic strategies. Cardiovascular diabetology 17: 121. https://doi.org/10.1186/s12933-018-0763-3.
Yin GN, Ryu JK, Kwon MH, Shin SH, Jin HR, Song KM, Choi MJ, Kang DY, Kim WJ, Suh JK (2012) Matrigel-based sprouting endothelial cell culture system from mouse corpus cavernosum is potentially useful for the study of endothelial and erectile dysfunction related to high-glucose exposure. The journal of sexual medicine 9: 1760–1772. https://doi.org/10.1111/j.1743-6109.2012.02752.x.
Wan Y, Liu Z, Wu A, Khan AH, Zhu Y, Ding S, Li X, Zhao Y, Dai X, Zhou J, Liu J, Li Y, Gong X, Liu M, Tian XL (2022) Hyperglycemia Promotes Endothelial Cell Senescence through AQR/PLAU Signaling Axis. Int J Mol Sci 23:2879. https://doi: 10.3390/ijms23052879.
Sun D, Chen S, Li S, Wang N, Zhang S, Xu L, Zhu S, Li H, Gu Q, Xu X, Wei F (2023) Enhancement of glycolysis-dependent DNA repair regulated by FOXO1 knockdown via PFKFB3 attenuates hyperglycemia-induced endothelial oxidative stress injury. Redox Biol 59: 102589. https://doi:10.1016/j.redox.2022.102589.
Shosha E, Xu Z, Narayanan SP, Lemtalsi T, Fouda AY, Rojas M, Xing J, Fulton D, Caldwell RW, Caldwell RB (2018) Mechanisms of diabetes-induced endothelial cell senescence: role of arginase 1. Int J Mol Sci 19: 1215. https://doi:10.3390/ijms19041215.
Xu S, Ilyas I, Little PJ, Li H, Kamato D, Zheng X, Luo S, Li Z, Liu P, Han J, Harding IC, Ebong EE, Cameron SJ, Stewart AG, Weng J (2021) Endothelial Dysfunction in Atherosclerotic Cardiovascular Diseases and Beyond: From Mechanism to Pharmacotherapies. Pharmacol Rev 73:924-967. https://doi:10.1124/pharmrev.120.000096.
Kim GD (2019) SIRT1-Mediated Protective Effect of Aralia elata (Miq.) Seem against High-Glucose-Induced Senescence in Human Umbilical Vein Endothelial Cells. Nutrients 11: 2625. https://doi:10.3390/nu11112625.
Bogdanović O, Delfino-Machín M, Nicolás-Pérez M, Gavilán MP, Gago-Rodrigues I, Fernández-Miñán A, Lillo C, Ríos RM, Wittbrodt J, Martínez-Morales JR (2012) Numb/Numbl-Opo antagonism controls retinal epithelium morphogenesis by regulating integrin endocytosis. Dev Cell 23: 782-795. https://doi:10.1016/j.devcel.2012.09.004.
Tagashira T, Fukuda T, Miyata M, Nakamura K, Fujita H, Takai Y, Hirata KI, Rikitake Y (2018) Afadin Facilitates Vascular Endothelial Growth Factor-Induced Network Formation and Migration of Vascular Endothelial Cells by Inactivating Rho-Associated Kinase Through ArhGAP29. Arterioscler Thromb Vasc Biol 38: 1159-1169. https://doi:10.1161/ATVBAHA.118.310991.
Xu H, Pumiglia K, LaFlamme SE (2020) Laminin-511 and α6 integrins regulate the expression of CXCR4 to promote endothelial morphogenesis. J Cell Sci 133: jcs246595. https://doi:10.1242/jcs.246595.
Zhou X, Stuart A, Dettin LE, Rodriguez G, Hoel B, Gallicano GI (2004) Desmoplakin is required for microvascular tube formation in culture. J Cell Sci 117: 3129-3140. https://doi:10.1242/jcs.01132.
Lu Q, Xie Z, Yan C, Ding Y, Ma Z, Wu S, Qiu Y, Cossette SM, Bordas M, Ramchandran R, Zou MH (2019) SNRK (Sucrose Nonfermenting 1-Related Kinase) Promotes Angiogenesis In Vivo. Arterioscler Thromb Vasc Biol 38: 373-385. https://doi:10.1161/ATVBAHA.117.309834.
Machado-Pereira M, Saraiva C, Bernardino L, Cristóvão AC, Ferreira R (2022) Argonaute-2 protects the neurovascular unit from damage caused by systemic inflammation. J Neuroinflammation 19: 11. https://doi: 10.1186/s12974-021-02324-7.
Azam SH, Porrello A, Harrison EB, Leslie PL, Liu X, Waugh TA, Belanger A, Mangala LS, Lopez-Berestein G, Wilson HL, McCann JV, Kim WY, Sood AK, Liu J, Dudley AC, Pecot CV (2019) Quaking orchestrates a post-transcriptional regulatory network of endothelial cell cycle progression critical to angiogenesis and metastasis. Oncogene 38: 5191-5210. https://doi:10.1038/s41388-019-0786-6.
Izumi N, Helker C, Ehling M, Behrens A, Herzog W, Adams RH (2012) Fbxw7 controls angiogenesis by regulating endothelial Notch activity. PLoS One 7: e41116. https://doi:10.1371/journal.pone.0041116.
Mammoto A, Muyleart M, Kadlec A, Gutterman D, Mammoto T (2018) YAP1-TEAD1 signaling controls angiogenesis and mitochondrial biogenesis through PGC1α. Microvasc Res 119: 73-83. https://doi:10.1016/j.mvr.2018.04.003.
Hogan BM, Bos FL, Bussmann J, Witte M, Chi NC, Duckers HJ, Schulte-Merker S (2009) Ccbe1 is required for embryonic lymphangiogenesis and venous sprouting. Nat Genet 41: 396-398. https://doi:10.1038/ng.321.
Jeltsch M, Jha SK, Tvorogov D, Anisimov A, Leppänen VM, Holopainen T, Kivelä R, Ortega S, Kärpanen T, Alitalo K (2014) CCBE1 enhances lymphangiogenesis via A disintegrin and metalloprotease with thrombospondin motifs-3-mediated vascular endothelial growth factor-C activation. Circulation 129: 1962-1971. https://doi:10.1161/CIRCULATIONAHA.113.002779.
Zou J, Wang N, Liu M, Bai Y, Wang H, Liu K, Zhang H, Xiao X, Wang K (2018) Nucleolin mediated pro-angiogenic role of Hydroxysafflor Yellow A in ischaemic cardiac dysfunction: Post-transcriptional regulation of VEGF-A and MMP-9. J Cell Mol Med 22: 2692-2705. https://doi:10.1111/jcmm.13552.
Hu C, Zhang X, Teng T, Ma ZG, Tang QZ (2022) Cellular Senescence in Cardiovascular Diseases: A Systematic Review. Aging Dis 13: 103-128. https://doi:10.14336/AD.2021.0927.
Laina A, Stellos K, Stamatelopoulos K （2018） Vascular ageing: Underlying mechanisms and clinical implications. Exp Gerontol 109: 16-30. https://doi:10.1016/j.exger.2017.06.007.
Mannell H, Hammitzsch A, Mettler R, Pohl U, Krötz F (2010) Suppression of DNA-PKcs enhances FGF-2 dependent human endothelial cell proliferation via negative regulation of Akt. Cell Signal 22: 88-96. https://doi:10.1016/j.cellsig.2009.09.015.
Chung JH (2018) The role of DNA-PK in aging and energy metabolism. FEBS J 285: 1959-1972. https://doi:10.1111/febs.14410.
Khor ES, Wong PF (2018) Endothelial replicative senescence delayed by the inhibition of MTORC1 signaling involves MicroRNA-107. Int J Biochem Cell Biol 101: 64-73. https://doi:10.1016/j.biocel.2018.05.016.
Xu X, Shen X, Feng W, Yang D, Jin L, Wang J, Wang M, Ting Z, Xue F, Zhang J, Meng C, Chen R, Zheng X, Du L, Xuan L, Wang Y, Xie T, Huang Z (2020) D-galactose induces senescence of glioblastoma cells through YAP-CDK6 pathway. Aging (Albany NY) 12:18501-18521. https://doi:10.18632/aging.103819.
Sheikh BN, Phipson B, El-Saafin F, Vanyai HK, Downer NL, Bird MJ, Kueh AJ, May RE, Smyth GK, Voss AK, Thomas T (2015) MOZ (MYST3, KAT6A) inhibits senescence via the INK4A-ARF pathway. Oncogene 34: 5807-5820. https://doi:10.1038/onc.2015.33.
Raynard C, Tessier N, Huna A, Warnier M, Flaman JM, Van Coppenolle F, Ducreux S, Martin N, Bernard D (2022) Expression of the Calcium-Binding Protein CALB1 Is Induced and Controls Intracellular Ca²⁺ Levels in Senescent Cells. Int J Mol Sci 23: 9376. https://doi:10.3390/ijms23169376.
Ruiz L, Traskine M, Ferrer I, Castro E, Leal JF, Kaufman M, Carnero A (2008) Characterization of the p53 response to oncogene-induced senescence. PLoS One 3: e3230. https://doi:10.1371/journal.pone.0003230.
Salminen A, Kaarniranta K (2011) Control of p53 and NF-κB signaling by WIP1 and MIF: role in cellular senescence and organismal aging. Cell Signal 23: 747-752. https://doi:10.1016/j.cellsig.2010.10.012.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

RNA-sequencing analysis reveals the potential mechanism by which a Dioscorea polystachya (Chinese yam) protein ameliorates hyperglycemia-induced endothelial dysfunction

Status:

Version 1

Abstract

Background

Methods and results

Conclusion

Figures

Introduction

Materials and methods

DP1 preparation

Cell culture

Cell viability assay

NO level determination

Tube formation assay

Senescence-associated β-galactosidase (SA-β-gal) staining

RNA extraction and sequencing

RNA-seq data analysis

Function and pathway enrichment analysis

RT-PCR validation of RNA-seq data

Statistical analysis

Results

DP1 enhanced cell viability and endothelial function in HUVECs exposed to HG conditions

DP1 prevented cell senescence in HUVECs exposed to HG conditions

Statistical summary of the RNA-seq data

Identification of DEGs

Functional and pathway enrichment analysis

DP1 increased the expression levels of DEGs involved in angiogenesis

DP1 treatment alters the expression levels of DEGs involved in cellular senescence

RT-PCR validation of RNA-seq data

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1