Study On The Mechanism Of Reducing Toxicity Of Triptolide Combined With Ferulic Acid Based On HaCaT Cells

Tripterygium is currently recognized as the most effective singl-dose Traditional Chinese medicine in the treatment of RA.However,oral preparations about Tripterygium have serious side effects,including hepatorenotoxicity,gastrointestinal and reproductive system toxicity,etc.These factors limit the clinical application of oral preparations.The external preparations are the commonly used way of administration in the treatment of rheumatoid arthritis.Because they have the advantages of direct action on target organs,rapid treatment and less toxic and side effects.Some studies have shown that the therapeutic effect of Tripterygium on RA is similar to that of oral administration,but the toxicity and side-effects are relatively lower after external administration.Triptolide is the main active component and quality-related component of Tripterygium preparations,and it is also the main toxic component.The previous pharmacodynamic study showed that Triptolide could engender certain toxicity after being administered through skin alone.The therapeutic effect of Triptolide combined with Ferulic acid on RA was similar to that of single administration,but the skin toxicity was decreased.However,there is no further study on the mechanism of reducing skin irritation.In this paper,we will explore the mechanism of Triptolide combined with Ferulic acid in reducing skin toxicity from the aspects of skin metabolism and oxidative damage by culturing epidermal cells in vitro.The aim is to provide a theoretical basis for the rational clinical application of TP and the development of external preparations.The main contents of this paper are as follows: 1.Selection of compatibility ratio of Triptolide and Ferulic acidThe survival rate of HaCaT cells was studied by MTT colorimetry,when the ratio of Triptolide and Ferulic acid was respectively 1:12.5,1:25,1:50,1:100 and 1: 120,and the drug’s action time was respectively 6 h,12 h and 24 h.When the survival rate arrived at 90%,which was the security line.The compatibility ratio of the two drugs was determined when the survival rate of the survival cells was the highest.The results showed that Triptolide had a serious toxic effect on HaCaT cells in a concentration and time dependent manner.With the increase of the drug concentration and the prolongation of the action time,the inhibitory effect of Triptolide on cell growth was significantly increased.Ferulic acid had no obvious effect on the growth of HaCaT cells in a certain concentration range,but the high concentration of Ferulic acid inhibited the growth of HaCaT cells.When the combination of Triptolide and Ferulic acid was used,ferulic acid could reduce the toxic effect of triptolide on HaCaT cells.When the compatibility ratio of triptolide and ferulic acid was 1:100 and the administration time was 6h,the effect of reducing toxicity was the best,and the range of non-toxic concentration of TP was increased by 7 times.This result was consistent with the best compatibility ratio by MDCK cell screening in the previous study of our research group.Finally,the compatibility ratio of triptolide and ferulic acid was determined to be 1:100,and the drug action time to HaCaT cells was 6 hours for following experiment.2.Effects of Ferulic acid on metabolic characteristics of Triptolide in HaCaT cell modelThe contents of Triptolide and Ferulic acid were determined by LC-MS/MS and HPLC,respectively.It was found that the metabolic transformation of TP increased within 6 hours after being used in combination,and there was significant difference between the high dose groups.The metabolic amount of TP in the low dose group was also higher than that in the control group,but there was no significant difference.In terms of the ability of cells to ingest drugs,the drug retention in the cells of each compatibility group was lower than that of TP alone group,however,there was no significant difference.The results indicated that Ferulic acid could accelerate the metabolism of Triptolide in HaCaT cells to a certain extent.The effects of Triptolide combined with Ferulic acid on the activity of CYP1A2,CYP2E1 and CYP3A4 in HaCaT cells were studied by "cocktail" probe with Fenacetine,Chlorzoxazone and Testosterone as probe substrates respectively.In a certain concentration range,Triptolide had a weak inhibitory effect on the activity of CYP2E1.There was no significant difference in enzyme activity between the TP single administration group with the each compatibility group.However,the results showed that Triptolide combined with Ferulic acid had no significant effect on the activities of CYP1A2 and CYP3A4.3.Protective effect of Triptolide combined with Ferulic acid on oxidative damage of HaCaT cellsThe activities of GSH,CAT,SOD and MDA in HaCaT cells were measured by enzyme biochemical method.The expression level of ROS was also detected by using 2,7 dichlorofuorescin diacetate(DCFH-DA,2,7-dichlorofuorescin diacetate)as probe.The results showed that the expression of ROS in high,middle and low dose groups of TP was significantly higher than that in normal cells,and showed a concentration-dependent manner on the whole.However,the content of ROS decreased obviously in each group when TP and ferulic acid were used in combination.ROS reached the same range as that of normal cells and maintained the redox homeostasis of HaCaT cells.TP can also significantly reduce the expression of GSH and CAT,and increase the activity of MDA and SOD in HaCaT cells.Triptolide combined with Ferulic acid could reverse regulate the oxidative damage of TP.The activity of GSH and CAT in HaCaT cells increased obviously,and the contents of MDA and SOD decreased,which made the oxidation factors reach the normal steady state level.In conclusion,Ferulic acid can repair redox equilibrium environment by damaged and improve the anti-oxidation ability of HaCaT cells,which through reducing the production of ROS and increasing the antioxidant components such as GSH and CAT,etc.The oxidative damage induced by Triptolide was further inhibited.4.Effects of Triptolide combined with Ferulic acid on the expression of Nrf2 proteinThe effect of Triptolide combined with Ferulic acid on the expression of Nrf2 protein in HaCaT cells was determined by western blotting.It was found that TP could decrease the expression of Nrf2 in HaCaT cells,but the effect was weak.However,Triptolide combined with Ferulic acid significantly increased the expression level of Nrf2,suggesting that ferulic acid may play a protective role in TP-induced oxidative damage by increasing the activity of Nrf2 and inhibiting the oxidative stress response.In conclusion,the combination of Triptolide and Ferulic acid may decrease the expression of ROS in HaCaT cells and promote Nrf2 activation by increasing TP metabolism,then increase the expression of key antioxidant factors such as GSH and CAT,and increase the antioxidant capacity of the epidermis.Thus,the skin irritation induced by Triptolide was reduced.