Platelet-rich plasma plays an antibacterial, anti-inflammatory and cell proliferation-promoting role in an in vitro model for diabetic infected wounds

Figures & data

Table 1 Components of the four co-culture groups

Group	H	HP	HS	HSP
Component
DMEM	1.6 mL	1.6 mL	1.4 mL	1.4 mL
S. aureusa	–	–	0.2 mL	0.2 mL
EPG	–	0.4 mL	–	0.4 mL
PPP	0.4 mL		0.4 mL	–

Notes:

a Staphylococcus aureus suspension was diluted with high glucose DMEM to 10,000/mL. The H group, culture of HaCaT cells alone; the HP group, uninfected HaCaT cells interfered with EPG; the HS group, co-culture of HaCaT cells with S. aureus; and the HSP group, S. aureus-infected HaCaT cells interfered with EPG.

Abbreviations: EPG, extract liquid of platelet-rich gel; PPP, platelet-poor plasma; S. aureus, Staphylococcus aureus.

Table 2 Primers used in the study

Primers	Sequences (5′→3′)
Reverse transcription PCR
U6	AAAATATGGAACGCTTCACG
miR-21	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCAACATC
Primers used for RT-qPCR
U6	Forward	CGCTTCGGCAGCACATATACTAAAATTGGAAC
	Reverse	GCTTCACGAATTTGCGTGTCATCCTTGC
miR-21	Forward	ACACTCCAGCTGGGTAGCTTATCAGACTGA
	Reverse	TGGTGTCGTGGAGTCG
IL-6	Forward	ACTCACCTCTTCAGAACGAATTG
	Reverse	CCATCTTTGGAAGGTTCAGGTTG
IL-10	Forward	TCAAGGCGCATGTGAACTCC
	Reverse	GATGTCAAACTCACTCATGGCT
TNF-α	Forward	CCTCTCTCTAATCAGCCCTCTG
	Reverse	GAGGACCTGGGAGTAGATGAG
TGF-β1	Forward	GGCCAGATCCTGTCCAAGC
	Reverse	GTGGGTTTCCACCATTAGCAC
β-actin	Forward	CTTAGTTGCGTTACACCCTTTCTT
	Reverse	CACCTTCACCGTTCCAGTTTT

Abbreviation: TNF-α, tumor-necrosis factor alpha.

Figure 1 Incubation of HaCaT cells with increasing starting concentrations of Staphylococcus aureus led to a dose-dependent decline of the cell proliferation.

Notes: Significant deviations from the HaCaT control at the respective incubation time (*P<0.05; **P<0.01).

Figure 2 Comparison of antibacterial activity between PRG and EPG and antibacterial performance of EPG at different concentrations.

Notes: (A) Comparison of the effects of PRG and EPG on the growth of S. aureus. (B) Effects of different concentrations of EPG on the growth of extracellular S. aureus. (C) Effects of different concentrations of EPG on the growth of intracellular S. aureus. Viable bacteria were quantified by the colony plate counting method. Significant deviations from the 30% PPP group (*P<0.05; **P<0.01).

Abbreviations: EPG, extract liquid of platelet-rich gel; PPP, Platelet-poor plasma; PRG, platelet-rich gel; S. aureus, Staphylococcus aureus.

Figure 3 Amounts of 10% and 20% EPG were tested for their capacity to protect HaCaT cells from bacterial damage and promote HaCaT cells proliferation.

Notes: Significant deviations from the HaCaT control at the respective incubation time (*P<0.05; **P<0.01).

Abbreviation: EPG, extract liquid of platelet-rich gel.

Figure 4 Images of the four groups under light microscope.

Notes: Photos were taken before extraction of RNA from HaCaT cells at various time points. Scale bar: 100 µm.

Figure 5 miR-21, PDCD4 and phospho-NF-κB p65 (P-P65) level in HaCaT cells of the four groups.

Notes: PDCD4 and p-p65 increased because of bacterial infection but decreased by EPG at the 12th and 24th hour. Direct application of EPG to HaCaT cells down-regulated PDCD4 and p-p65 protein at the 24th and 48th hour. MiR-21 elevated with reduced PDCD4 and p-p65 at the 24th and 48th hour. In the setting of bacterial infection, results at the 48th hour are not shown because of insufficient RNA and protein extracted from the residual cells. Significant deviations from the HaCaT control at the respective incubation time (*P<0.05; **P<0.01).

Abbreviations: EPG, extract liquid of platelet-rich gel; PDCD4, programmed cell death factor 4.

Figure 6 Immunofluorescence microscopy analysis of the spatial localization of the NF-κB p65 protein.

Figure 7 (A) IL-6, (B) IL-10, (C) TNF-α, (D) TGF-β1 mRNA expression in HaCaT cells as measured by qRT-PCR.

Notes: In the setting of bacterial infection, results at the 48th hour are not shown because of insufficient RNA extracted from residual cells. Significant deviations from the HaCaT control at the respective incubation time (*P<0.05; **P<0.01).

Abbreviations: EPG, extract liquid of platelet-rich gel; qRT-PCR, quantitative reverse transcription; TNF-α, tumor-necrosis factor alpha.

Figure 8 (A) IL-6, (B) IL-10, (C) TNF-α, (D) TGF-β1 content in the supernatant as determined by ELISA.

Notes: Significant deviations from the HaCaT control at the respective incubation time (*P<0.05; **P<0.01).

Abbreviations: EPG, extract liquid of platelet-rich gel; TNF-α, tumor-necrosis factor alpha.