The Study On The Promoting Penetration Mechanism Of Menthol Involving The PLC Activity Of Keratinocyte

ObjectiveTo study the promoting penetration mechanism of menthol to transdermal drugsby means of investigating the relationship among the level of free intracellularcalcium, the activity of phospholipase C（PLC） and the change of skin structure.Methods1. In vitro skin penetration experimentUsing modified Franz diffusion cell device in vitro, selecting the rabbit back skinas transdermal barrier and taking acetaminophen as model drug, the promotingpenetration effect of different penetration enhancers, whose concentration was thesame, were evaluated by some indicators including cumulative permeation amount,the steady-state flux and the lag time.2. Calcium ion assay experimentAfter split charging, the keratinocytes were adding different culture mediaaccording to the groups which contained1‰DMSO,50,100,200μM menthol, PLCinhibitor （U73122）and nifedipine. By using flow cytometer bound with Fluo-3/AMstaining technology, the effect of menthol on the free intracellular calcium level ofkeratiioncytes.3. PLC activity test experimentCultured keratinocytes were incubated for4hour in different culture mediawhich contained1‰DMSO,50,100,200μM menthol and PLC inhibitor（U73122）. Then, according to the directions, the PLC activity of keratinocytes were tested byPLC activity ELISA kit. After incubating200μM menthol for4h. the normal culturemedia was take place of the old one, and the keratinocytes were tested by the test kitat interval of2,4,8,12,24hour.4. Ca²⁺-ATPase activity test experimentCultured keratinocytes were incubated for4hour in different culture mediawhich contained1‰DMSO,50,100,200μM menthol and PLC inhibitor（U73122）.Then, according to the direction, the Ca²⁺-ATPase activity of keratinocytes weretested by Ca²⁺-ATPase activity test kit.5. ATP assay test experimentCultured keratinocytes were incubated for4hour in different culture mediawhich contained1‰DMSO,50,100,200μM menthol and PLC inhibitor（U73122）.Then, according to the direction, the ATP content of keratinocytes were tested byATP assay test kit.6. PKC activity test experimentCultured keratinocytes were incubated for4hour in different culture mediawhich contained1‰DMSO,50,100,200μM menthol、PLC inhibitor（U73122） andprotein kinase C（PKC） inhibitor （chelerythrine）. Then, according to the directions, thePKC activity of keratinocytes were tested by PKC activity ELISA kit. Afterincubating200μM menthol for4h. the normal culture media was take place of the old,and the keratinocytes were tested by the test kit at interval of2,4,8,12,24hour.7. Membrane fluidity test experimentAfter more than90%cells attaching on the culture dish, keratinocytes wereincubated for4hour in different culture media which contained1‰DMSO,50,100,200μM menthol and PKC inhibitor（chelerythrine）. Then, the keratinocytes wereincubated with1,6-Diphenyl-1,3,5-hexatriene（DPH） for30min in37℃. Thefluorescence intensity of keratinocytes were detected using a fluorescencespectrophotometer contained a set of polarization device（Excitation wavelength: 362nm, Emission wavelength:432nm）, and the fluorescence polarization andmicroviscosity of keratinocytes were calculated by mathematics.8. Microfilament skeleton staining experimentInoculated keratinocytes into six-well plates culture dish which has lieda cover glass. After attaching on the glass, keratinocytes were incubated for4hour in different culture media which contained1‰DMSO,50,100,200μM menthol and PKC inhibitor（chelerythrine）. Then the microfilament skeleton of keratinocytes were stained by Coomassie brilliant blue and it was examined undermicroscope.Results1The evaluation on promoting penetration effect of different penetrationenhancersCompared with the cumulative permeation amount of blank group, propanediolgroup and camphor group were increased （p<0.05）, and azone group, menthol groupand oleic acid group were increased sharply（p<0.01）. Compared with the cumulativepermeation amount of azone group, oleic acid group, propanediol group and camphorgroup were less（p<0.01）. Compared with the steady-state flux of blank group,propanediol group and camphor group were increased （p<0.05）, and azone group,menthol group and oleic acid group were increased sharply（p<0.01）. Compared withthe steady-state flux of azone group, oleic acid group had no significant difference（p>0.05）, menthol group was more（p<0.05）but propanediol group and camphor groupwere less（p<0.01）. Compared with the steady-state flux of azone group, oleic acidgroup had no significant difference（p>0.05）, menthol group was more （p<0.05） butpropanediol group and camphor group were less（p<0.01）. Compared with the lag timeof blank group, azone group, menthol group and propanediol group had no significantdifference（p>0.05）, camphor group was shorter （p<0.05）, oleic acid group werelonger obviously （p<0.01）. Compared with the lag time of azone group, mentholgroup and camphor group had no significant difference（p>0.05）, propanediol groupwas longer（p<0.05） and camphor group was also longer （p<0.01）. 2. The influence on the free intracellular calcium ion of keratinocytes by mentholCompared with the fluorescence intensity of blank group, DMSO group, U73122group and nifedipine group had no significant difference（p>0.05）, and fluorescenceintensity was increased with the concentration of menthol （p<0.01）. The fluorescenceintensity of menthol+U71322group was lower than the same concentration mentholgroup （p<0.01） but more than blank group （p<0.01）.3. The influence on the PLC activity of keratinocytes by mentholCompared with the PLC activity of blank group, DMSO group had no significantdifference（p>0.05）, U73122group was lower （p<0.01）, and PLC activity wasincreased with the concentration of menthol （p<0.01）. The PLC activity ofmenthol+U71322group was lower than the same concentration menthol group（p<0.01） and had no significant difference with blank group（p>0.05）. After instead ofnormal culture media, the PLC activity of200μM menthol group was recovery slowlyin time.4. The influence on the Ca²⁺-ATPase activity of keratinocytes by mentholCompared with the Ca²⁺-ATPase activity of blank group, DMSO group wasreduced（p<0.05）but U73122group was increased（p<0.05）, and Ca²⁺-ATPase activitywas reduced with the concentration of menthol （p<0.01）. The Ca²⁺-ATPase activity ofmenthol+U71322group was more than the same concentration menthol group（p<0.05） but still less than blank group （p<0.01）.5. The influence on the ATP assay of keratinocytes by mentholCompared with the ATP level of blank group, DMSO group had no significantdifference（p>0.05）, U73122group was increased（p<0.05）, and ATP level wasreduced with the concentration of menthol （p<0.01）. The ATP level of menthol+U71322group was more than the same concentration menthol group （p<0.05） but stillless than blank group（p<0.01）.6. The influence on the PKC activity of keratinocytes by menthol Compared with the PKC activity of blank group, DMSO group had nosignificant difference（p>0.05）, U73122group was lower（p<0.05）, chelerythrine groupwas also lower（p<0.01）, and PKC activity was increased with the concentration ofmenthol（p<0.01）. The PKC activity of menthol+U71322group was lower than thesame concentration menthol group （p<0.01） but still more than blank group（p<0.01）.After instead of normal culture media, the PKC activity of200μM menthol group wasrecovery slowly in time.7. The influence on the membrane fluidity of keratinocytes by mentholCompared with the fluorescence polarization of blank group, DMSO group andchelerythrine group had no significant difference （p>0.05）,50μM menthol groupwas reduced （p<0.05）,100μM menthol group,200μM menthol group and menthol+chelerythrine group was reduced sharply （p<0.01）. The fluorescence polarization ofmenthol+chelerythrine group was lower than the same concentration menthol group（p<0.05） but still more than blank group（p<0.01）. Compared with the microviscosityof blank group, DMSO group and chelerythrine group had no significantdifference（p>0.05）, All of menthol group was reduced（p<0.01）, menthol+chelerythrine group was reduced sharply （p<0.01）. The fluorescence polarization ofmenthol+chelerythrine group was lower than the same concentration menthol group（p<0.05） and blank group（p<0.01）.8. The influence on the microfilament skeleton of keratinocytes by mentholCompared with blank group, DMSO group and chelerythrine group had nodifferences. But the volume of keratinocytes with menthol was reduced and the celljunction became loose and intercellular space was increased. Compared with mentholgroup, the intercellular space of menthol+chelerythrine group was reduced obviouslyand cell junction was tighter. However, the improvement of menthol+chelerythrinegroup still have some differences to blank group.Conclusion1. Even though they are the same concentration, different enhancers, have different promoting penetration effect to identical transdermal drug.2. Menthol could activate the calcium ion channel of keratinocytes to increasecalcium ion by mean of increasing PLC activity.3. Menthol could reduce Ca²⁺-ATPase activity of keratinocytes to increase calciumion by mean of increasing PLC activity.4. Menthol could reduce ATP level of keratinocytes involving PLC activity.5. PKC activity of keratinocytes is increased because of the increase of PLCactivity induced by menthol.6. PKC is playing an negative regulation in the increase of membrane fluidityinduced by PKC.7. Menthol could change microfilament cytoskeleton of keratinocytes involvingPKC activity.