Necrostatin-1 promotes the proliferation and osteogenic differentiation of periodontal ligament stem cells in high-glucose environment

doi:10.3877/cma.j.issn.1674-1366.2022.03.005

Abstract

Abstract:

Objective

To investigate the effect of necrostatin-1 (Nec-1) on the biological characteristics of periodontal ligament stem cells (PDLSCs) in the high-glucose environments in vitro.

Methods

PDLSCs were successfully cultured by single colony and divided into three groups according to the following treatments: control group (5 mmol/L glucose) , high-glucose group (25 mmol/L glucose) , high-glucose + Nec-1 group (25 mmol/L glucose + 30 μmol/L Nec-1) . Western blot was used to detect the expression of necroptosis related molecules (RIP1 and RIP3) and the cell proliferation of PDLSCs were evaluated by MTT assay in the above three groups. The osteogenic differentiation of PDLSCs were evaluated by alizarin red staining, the quantitative detection of alkaline phosphatase (ALP) and real-time quantitative PCR assay. All statistical analyses were used SPSS 26.0 software. One way ANOVA was used to compare the cell proliferation activity (A value of the MTT absorbance) , the expression of ALP and the relative levels of osteogenesis related genes (COL1, RUNX2, OCN) among the groups, and the LSD-t test was used for multiple comparisons between the groups.

Results

Western blot showed that the expression levels of RIP1 and RIP3 in high-glucose group were significantly higher than those in control group, while significantly lower in high-glucose + Nec-1 group than in high-glucose group. The proliferation activity of PDLSCs at day 7 in high-glucose group was significantly lower than that in the control group, and its MTT absorbance (0.67 ± 0.06) was significantly lower than that of the control group (1.23 ± 0.12) (t = 9.652, P<0.001) . After the inhibition of Nec-1, the proliferation activity of PDLSCs increased significantly, and the MTT absorbance at day 7 of high-glucose + Nec-1 group (1.12±0.11) was significantly higher than that of high-glucose group (0.67±0.06) (t = 8.185, P<0.001) . Compared with the control group, the mineralized nodules formed by PDLSCs at day 14, the levels of ALP (3.42 ± 0.37) and the relative expression of osteogenesis related genes COL1 (1.86 ± 0.16) , RUNX2 (1.55 ± 0.23) , OCN (1.08 ± 0.20) at day 7 were significantly lower in high-glucose group (t_ALP = 13.149, t_COL1 = 14.257, t_RUNX2 = 7.593, t_OCN = 8.606, all P<0.001) . After the inhibition of Nec-1, the osteogenic differentiation of PDLSCs increased, and the mineralized nodules formed by PDLSCs at day 14, the levels of ALP (6.06±0.26) and the relative expression of osteogenesis related genes COL1 (3.64 ± 0.30) , RUNX2 (2.53 ± 0.26) , OCN (2.14 ± 0.30) at day 7 of high-glucose + Nec-1 group were significantly higher than those of high-glucose group (t_ALP = 13.033, t_COL1 = 11.636, t_RUNX2 = 6.332, t_OCN = 6.573, all P<0.001) .

Conclusions

The proliferation and osteogenic differentiation of PDLSCs were inhibited in the high-glucose environments in vitro. Nec-1 significantly improved the proliferation and osteogenic differentiation of PDLSCs in the high-glucose environments.

Key words: Necrostatin-1, Periodontal ligament, Stem cells, Biological characteristic, Osteogenic differentiation

Chenglin Liu, Wenxing Xun, Haizhen Yang, Sumeng Fan, Yubo Liu, Hongmei Zhang. Necrostatin-1 promotes the proliferation and osteogenic differentiation of periodontal ligament stem cells in high-glucose environment[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2022, 16(03): 160-167.

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Figures/Tables 11

References 20

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目的基因	引物序列	产物长度（bp）	基因库登记号
COL1	F：5′-GGGCGAGTGCTGTGCTTT-3′	180	NM_000089.4
	R：5′-GACCCATTGGACCTGAACC-3′
RUNX2	F：5′-CACTGGCGCTGCAACAAGA-3′	176	NM_001015051.4
	R：5′-ACTTGGTTTTTCATAACAGCGGA-3′
OCN	F：5′-ATGAGGACCCTCTCTCTGCTC-3′	155	NM_199173.6
	R：5′-CTAAACGGTGGTGCCATAGAT-3′
β-actin	F：5′-GAGACCTTCAACACCCCAGCC-3′	160	NM_001101.5
	R：5′-CATAGCACAGCTTCTCTTTAA-3′

目的基因	引物序列	产物长度（bp）	基因库登记号
COL1	F：5′-GGGCGAGTGCTGTGCTTT-3′	180	NM_000089.4
	R：5′-GACCCATTGGACCTGAACC-3′
RUNX2	F：5′-CACTGGCGCTGCAACAAGA-3′	176	NM_001015051.4
	R：5′-ACTTGGTTTTTCATAACAGCGGA-3′
OCN	F：5′-ATGAGGACCCTCTCTCTGCTC-3′	155	NM_199173.6
	R：5′-CTAAACGGTGGTGCCATAGAT-3′
β-actin	F：5′-GAGACCTTCAACACCCCAGCC-3′	160	NM_001101.5
	R：5′-CATAGCACAGCTTCTCTTTAA-3′

组别	样本量	第1天	第3天	第7天	F值	P值
对照组	5	0.28 ± 0.04	0.56 ± 0.05	1.23 ± 0.12	192.229	<0.001
高糖组	5	0.17 ± 0.05	0.32 ± 0.05	0.67 ± 0.06	116.245	<0.001
高糖+Nec-1组	5	0.25 ± 0.03	0.51 ± 0.06	1.12 ± 0.11	184.496	<0.001
F值		9.585	26.425	45.902
P值		0.003	<0.001	<0.001

组别	样本量	第1天	第3天	第7天	F值	P值
对照组	5	0.28 ± 0.04	0.56 ± 0.05	1.23 ± 0.12	192.229	<0.001
高糖组	5	0.17 ± 0.05	0.32 ± 0.05	0.67 ± 0.06	116.245	<0.001
高糖+Nec-1组	5	0.25 ± 0.03	0.51 ± 0.06	1.12 ± 0.11	184.496	<0.001
F值		9.585	26.425	45.902
P值		0.003	<0.001	<0.001

组别	样本量	第1天	第3天	第7天	F值	P值
对照组	5	1.71 ± 0.14	3.28 ± 0.33	6.25 ± 0.31	358.298	<0.001
高糖组	5	0.87 ± 0.17	1.80 ± 0.24	3.42 ± 0.37	112.192	<0.001
高糖+Nec-1组	5	1.31 ± 0.17	2.99 ± 0.39	6.06 ± 0.26	350.625	<0.001
F值		35.019	28.970	125.009
P值		<0.001	<0.001	<0.001

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