Puerarin transport across a Calu-3 cell monolayer – an in vitro model of nasal mucosa permeability and the influence of paeoniflorin and menthol

Figures & data

Figure 1 Chemical structures of (A) puerarin, (B) paeoniflorin, and (C) menthol.

Figure 2 Cytotoxicity of puerarin, paeoniflorin, menthol, and their combinations as assessed by MTT test in Calu-3 cells for 24 hours.

Notes: (A) Cytotoxicity of puerarin, (B) cytotoxicity of paeoniflorin, (C) cytotoxicity of menthol, (D) cytotoxicity of puerarin combined with paeoniflorin, (E) cytotoxicity of puerarin combined with menthol, and (F) cytotoxicity of puerarin combined with paeoniflorin and menthol. Data expressed as mean ± SD (n=5). *P<0.05 compared with the control group.
Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD, standard deviation.

Table 1 Transport of increasing puerarin concentrations across Calu-3 cell monolayers

Condition (μg/mL)	Papp (A→B) (×10^−6 cm/s)	Papp (B→A) (×10^−6 cm/s)	ER (B→A/A→B)
30	1.220±0.027	1.174±0.053	0.963±0.065
60	1.226±0.039	1.240±0.055	1.012±0.032
120	1.238±0.053	1.266±0.104	1.023±0.070

Notes: P_app, permeability; A, apical side; B, basolateral side. Values shown as mean ± SD (n=3).

Abbreviations: ER, efflux ratio; s, seconds; SD, standard deviation.

Table 2 Effects of paeoniflorin (Pa) and menthol (Me) on puerarin (Pu) transport in Calu-3 cells

Condition (μg/mL)	Papp (A→B) (×10^−6 cm/s)	Papp (B→A) (×10^−6 cm/s)	ER (B→A/A→B)
Pu (60) (control)	1.226±0.039	1.240±0.055	1.012±0.032
+Pa (12)	1.240±0.027	1.242±0.037	1.002±0.039
+Pa (24)	1.233±0.054	1.254±0.032	1.018±0.065
+Pa (48)	1.229±0.025	1.289±0.035	1.049±0.027
+Me (15)	1.285±0.084	1.363±0.083	1.065±0.116
+Me (30)	1.639±0.058*	1.803±0.111*	1.100±0.060
+Me (60)	1.788±0.087*	2.067±0.151*	1.157±0.088
+Pa (12) + Me (15)	1.305±0.150	1.288±0.067	0.999±0.150
+Pa (24) + Me (30)	1.559±0.143*	1.910±0.254*	1.228±0.157
+Pa (48) + Me (60)	1.957±0.067*	2.136±0.130*	1.091±0.048

Notes: P_app, permeability; A, apical side; B, basolateral side. Values shown as mean ± SD (n=3). Differs from Pu (60 μg/mL):

* P<0.05.

Abbreviations: ER, efflux ratio; s, seconds; SD, standard deviation.

Figure 3 The trend of TEER change in A→B and B→A transport in Calu-3 cells after treatment with puerarin, either alone or in combination with paeoniflorin and menthol.

Notes: (A) Exposure to different concentrations of puerarin, and no significant TEER change was observed compared with the control group. (B) Exposure to a specific amount of puerarin combined with different concentrations of paeoniflorin, and TEER also showed no significant change. (C) Exposure to a specific amount of puerarin combined with different concentrations of menthol, and TEER decreased significantly compared with the control group. (D) Exposure to a specific amount of puerarin combined with different concentrations of paeoniflorin and menthol, and TEER tended to decrease. The TEER at each time point is represented by the mean ± SD (n=3). Significant difference from TEER of the cells without treatment: *P<0.05.
Abbreviations: TEER, transepithelial electrical resistance; min, minutes; SD, standard deviation; P, puerarin; PP, puerarin and paeoniflorin; PM, puerarin and menthol; PPM; puerarin, paeoniflorin and menthol.

Figure 4 Effects of puerarin, paeoniflorin, and menthol on TJ proteins in Calu-3 cells.

Notes: Immunocytochemistry showed no differences in staining for the three types of TJ proteins in Calu-3 cells treated with puerarin and paeoniflorin compared with the control group, whereas the three TJ proteins showed decreased staining after menthol treatment. The scale bar corresponds to 15 μm.
Abbreviation: TJ, tight junction.

Figure 5 Changes to the relative AOD of occludin, claudin-1, and F-actin in Calu-3 cells after treatment with different compounds.

Notes: The relative AOD values for TJ proteins did not significantly differ after treatment with puerarin or paeoniflorin compared with the control group (P>0.05), whereas the values of the menthol group decreased significantly (P<0.05). Data shown as mean ± SD (n=3). *P<0.05 compared with the value in the control group.
Abbreviations: AOD, average optical density; TJ, tight junction; SD, standard deviation.