Anti-inflammatory effects of shikonin in human periodontal ligament cells

Figures & data

Figure 1. Chemical structure of shikonin (Zhao et al. 2017).

Table 1. Primers used in qRT-PCR.

Gene	Primers: Forward (F) and Reverse (R)	Annealing Temperature
IL-1	F: 5′-TTACAGTGGCAATGAGGATGAC-3′	60 °C
	R: 5′-TGCTGTAGTGGTGGTCGGAGA-3′
IL-6	F: 5′-AGGAGACTTGCCTGGTGAAA-3′	58 °C
	R: 5′-CAGGGGTGGTTATTGCATCT-3′
TNF-α	F: 5′-CCTGGTATGAGCCCATCTATC-3′	59 °C
	R: 5′-GGTTGGATGTTCGTCCTCCTC-3′
MMP-2	F: 5′-CCGTCGCCCATCATCAA-3′	58 °C
	R: 5′-AGATATTGCACTGCCAACTCT-3′
MMP-9	F: 5′-TCGTGGTTCCAACTCGGTTT-3′	61 °C
	R: 5′-GCGGCCCTCGAAGATGA-3′
COX-2	F: 5′- AGGCTTCCATTGACCAGAGC-3′	60 °C
	R: 5′- TCCACAGCATCGATGTCACC-3′
18s	F: 5′-TTCGGAACTGAGGCCATGAT-3′	60 °C
	R: 5′-CGAAC CTCCGACTTCGTTC-3′

Figure 2. Effect of SHI on hPDLC proliferation. hPDLCs were exposed to SHI (0.125, 0.25, 0.5, 1, and 2 μg/mL) for 1, 3 and 7 days. Cell proliferation was measured using the MTT assay. The data are expressed as the percentage of the control (containing medium only). Error bars indicate mean ± SEM (n = 3). *p < 0.05 versus the control. Statistical analysis was performed using One-way ANOVA and Tukey’s post hoc test.

Figure 3. Effect of SHI on hPDLC morphology. hPDLCs were pretreated with SHI (0.25 and 0.5 µg/mL) for 1 h and then stimulated with LPS for 12 h. Images were then captured using a Nikon Eclipse TS100 light microscope (A) and a Nikon Eclipse TE2000-U fluorescence microscope (B).

Figure 4. Effect of SHI on hPDLC gene expression. hPDLCs were pretreated with SHI (0.25 and 0.5 µg/mL) for 1 h and then stimulated with LPS for 1 and 3 days, after which total RNA was collected. After RNA extraction, first strand cDNA was synthesized. The cDNA sample was then amplified using qRT-PCR. The expression of the target gene was first normalized to 18s and then further converted to the percentage of the control. Error bars indicate mean ± SEM (n = 3). #p < 0.05 versus the control while *p < 0.05 versus the LPS group. Statistical analysis was performed using One-way ANOVA and Tukey’s post hoc test.

Figure 5. SHI-induced signaling pathways in hPDLCs. hPDLCs were pretreated with SHI (0.25 and 0.5 µg/mL) for 1 h and then stimulated with LPS for 30 and 60 min, after which, total protein was collected. The levels of protein expression were determined by western blotting. The expression of p-ERK was converted to the percentage of total ERK; expression of NF-κB and I-κB were converted to GAPDH. Then the data was further converted into the percentage of the control group. Error bars indicate mean ± SEM (n = 3). #p < 0.05 versus the control while *p < 0.05 versus the LPS group. Statistical analysis was performed using One-way ANOVA and Tukey’s post hoc test.

Zhao X, Zhou Y, Wang L, Li M, Shi D, Li D, Wang J. 2017. Shikonin alleviates the biotoxicity produced by pneumococcal pneumolysin. Life Sci. 177:1–7.

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