| Epidermal growth factor receptor inhibitors(EGFRIs)have been widely recognized due to their outstanding efficacy in the treatment of malignant tumors,such as non-small cell lung cancer and colorectal cancer,and now have many clinical applications,but their accompanying adverse effects also cause great distress to patients undergoing treatment.As the earliest and most frequent EGFRIs related cutaneous adverse effects,rash has a great impact on various aspects,such as patients' image,social as well as quality of life,and even caused some patients to stop receiving EGFRIs treatment which has huge impact on patients' survival.It is currently believed that the occurrence of EGFRIs related rash is associated with mechanisms such as cytokine mediated inflammatory response,so clinical guidelines recommend antibiotics and hormones to treat the rash,but the efficacy is unsatisfactory and antibiotics and hormones are similarly accompanied by lots of side effects.Seeking the methods of traditional Chinese medicine(TCM)to treat EGFRIs related rash becomes the hot spot of current research,Prof.Huijuan Cui's team subconsciously studied the TCM treatment of EGFRIs related rash for many years and developed a TCM compound external preparation,"Zhiyang Pingfu Decoction"(ZYPF),which achieved certain efficacy and good safety.However,the mechanism of ZYPF remains unclear and needs to be explored in-depth studiesIn this study,patients with moderate to severe rash associated with EGFRIs were enrolled to test the efficacy and safety of ZYPF,based on a randomized controlled trial.In parallel,the serum cytokine levels of the enrolled patients before and after treatment were examined to explore the mechanism of ZYPF.Qualitative and quantitative detection of ZYPF was performed using ultra performance liquid chromatography to identify the major active monomer in the compound,and exploration of the mechanism of this monomer on a TNF-?-mediated inflammation model of HaCaT cells was performed to provide experimental evidence for the clinical use of ZYPFPart ? Clinical trials1.ObjectiveThe efficacy and safety of short-term,low-dose oral minocycline±methylprednisolone tablets combined with ZYPF for the treatment of EGFRIs related rash were tested by a randomized,controlled clinical trial.The changes of serum cytokine levels before and after treatment in enrolled patients were also explored.2.MethodsThe patients with EGFRIs related rash were enrolled in a double-blind,randomized,controlled trial and randomly assigned to test and control groups.The patients in the test group received ZYPF combined with standard treatment of Western medicine,ZYPF topical twice daily,hydrocortisone butyrate cream topical twice daily,minocycline capsules oral twice daily 100 mg each time,patients with severe rash oral methylprednisolone tablets once daily 20 mg each time as necessary.Patients in the control group received placebo combined with standard treatment of Western medicine.Serum cytokine assays were performed after 7 days of treatment in all patients,and efficacy was evaluated after 7 and 14 days of treatment.The primary efficacy indicator was the treatment response rate of moderate to severe rash associated with EGFRIs,and secondary efficacy indicators included the itch symptom score accompanying rash,other cutaneous adverse effects grade of EGFRIs,minocycline and methylprednisolone dosage,and quality of life scores.Serum cytokine levels were compared before and after treatment,and safety indicators were monitored.3.ResultsA total of 58 patients were included,4 were dislodged,and a total of 54 patients completed the clinical trial for statistical analysis,including 27 in the test group and 27 in the control group.In terms of the primary efficacy indicator of the study,the treatment effective rate of the test group after 14 days of treatment was 81.48%,and the control group was 55.56%,and the difference was statistically significant(P=0.040).In terms of secondary efficacy indicators,after 14 days of treatment,the effective rates of the test group and the control group in the treatment of EGFRIs related skin pruritus were 100.00%and 82.61%,the difference between the two was statistically significant(P=0.033),the treatment effective rates of EGFRIs related skin xerosis were 80.77%and 70.37%,respectively,the difference was not statistically significant(P>0.05),and the treatment response rates of EGFRIs related paronychia were 76.92%and 68.42%,respectively,with no significant difference between them(P>0.05).The median duration of oral minocycline administration was 7 days in both the test and control groups,with a median total dose of 1000 mg in the test group and 1400 mg in the control group.Only 1 patient in the control group took methylprednisolone tablets.Quality of life scores improved significantly in both groups after treatment(P<0.001).After 7 days of treatment,the serum IL-8 level in the test group was lower than that in the control group,and the difference between the two was statistically significant(P=0.018).No statistical differences were found in the differences between the other cytokines(P>0.05).Four patients in the trial experienced gastrointestinal adverse effects,which improved after discontinuation of minocycline,and no adverse effects directly related to ZYPF occurred4.ConclusionThe long-term topical use of traditional Chinese medicine ZYPF combined with short-term,low-dose oral minocycline capsules ± methylprednisolone tablets showed better efficacy in the treatment of EGFRIs related moderate to severe rash,equally significant efficacy in the treatment of pruritus associated with rash,significant improvement in patients' quality of life,a lowering effect on patients' serum IL-8 levels,and no effect on other cytokines.The application of ZYPF can reduce the dosage of minocycline capsules and methylprednisolone tablets,reduce the side events brought by antibiotics and hormones,so that the therapeutic efficacy of rash is maintained for a long time and effectively,with low adverse effects.This study provides new evidence on the therapeutic mode of short-term oral standard treatment of Western medicine,combined with long-term topical ZYPF for the treatment of EGFRIs related rash,and brings a new treatment modality for rash in patients treated with long-term EGFRIsPart ? Basic experiments1.ObjectiveAfter searching for the main monomeric components of compound ZYPF by ultra-performance liquid chromatography,TNF-? was used to construct the inflammation model of HaCaT cells,and the main active monomers of ZYPF were applied to explore the anti-inflammatory effect based on PLA2G4A gene on TNF-?-mediated inflammation model of HaCaT cells.2.MethodsZYPF samples were analyzed by ultra-performance liquid chromatography,and the major monomeric components were identified by alignment with standards.20 ng/mL of TNF-? was applied to intervened HaCaT cells,CCK8 was applied for the detection of cell viability,PI-FACS for the detection of cell cycle,Annexin V-APC&PI double staining for the detection of apoptosis,Western blot for the detection of autophagy proteins,ELISA for the detection of IL-6 levels,for the assessment of TNF-? on HaCaT cells at different intervention times,such as 24 h,48 h and 72 h,meanwhile,the optimal action time of TNF-? was determined.The active monomers of ZYPF at different concentrations and minocycline at 5 mg/L as a positive control were used to intervene the HaCaT inflammatory cell model,and the cell activities were examined by CCK8,protein expression of PLA2G4A gene by Western blot,and IL-6 levels by ELISA3.ResultsFour major components,including matrine,baicalin,wogonoside and baicalein,were collectively recognized by comparison with the corresponding chromatographic peaks of control standards under the ultra-performance liquid chromatography conditions,among which matrine was the most abundant at a concentration of 15.565 mg/mL,and baicalin at a concentration of 4.160 mg/mL.After TNF-? intervention in HaCaT cells,an increase in cell absorbance was detected by CCK8 assay,with the greatest difference between the two groups at 72 h,and the difference between the two groups was statistically significant(P=0.008).After TNF-? intervention in HaCaT cells for 72 h,the proportion of apoptotic cells increased significantly compared with cells without TNF-? intervention(P<0.001),the number of HaCaT cells in G1 phase was significantly more than that in the control group,the difference between the two was statistically significant(P<0.001),the number of cells in G2/M and S phase was significantly less than that in the control group,the difference between the two was not statistically significant(P>0.05).After TNF-? intervention in HaCaT cells for 72 h,LC3B protein expression in HaCaT cells was significantly decreased(P<0.01),and there was a significant statistical difference in IL-6 levels between them compared with cells without TNF-? intervention(P<0.001),suggesting that the TNF-a-mediated inflammation model of HaCaT cells was successful when applied at 72 h.After application of minocycline and different concentrations of matrine to intervene HaCaT inflammatory cells,CCK8 assay found that the absorbance of cells in each group decreased compared with that before,but the difference with the TNF-a-mediated inflammation model group was not statistically significant(P>0.05).Compared with the inflammation model group,the levels of IL-6 in the minocycline group,the matrine 200 ?g/mL,400?g/mL and 800 ?g/mL groups were decreased,and the levels of IL-6 in the matrine 100?g/mL group were increased,but none of the differences with the model group were statistically significant(P>0.05).The inflammation model group showed a significant decrease in cPLA2 protein expression compared with the blank control group,and the difference between the two groups was statistically significant(P<0.001),after the intervention of minocycline and different concentrations of matrine,the cPLA2 protein expression compared with the model group all increased(P<0.05).cPLA2 protein expression in the matrine 100 ?g/mL,200 ?g/mL and 800 ?g/mL groups was significantly increased compared with that in the minocycline group(P<0.05),and the differences between the 400 ?g/mL group and minocycline group were not statistically significant(P>0.05).4.ConclusionAll the components of the detected chromatographic peaks in the ZYPF samples were water-soluble components under ultra-performance liquid chromatography conditions,and four major components could be identified by comparison with the reference standards,which were:matrine,baicalin,wogonoside and baicalein,with the highest content being matrine at a concentration of 15.565 mg/mL.Treatment of HaCaT cells with TNF-? for 72 h promoted proliferation and apoptosis of HaCaT cells,inhibited autophagy of HaCaT cells,had an effect on the cell cycle of HaCaT cells,promoted the increase of IL-6 expression level in HaCaT cells,and succeeded in modeling inflammation of HaCaT cells.Matrine and minocycline could inhibit the proliferation of HaCaT inflammatory cells mediated by TNF-? and decrease the expression level of IL-6 in inflammatory cells,and matrine was superior to minocycline.In TNF-?-mediated HaCaT inflammatory cells,cPLA2 protein expression was significantly decreased,which could be enhanced by minocycline and matrine,with matrine outperforming minocycline.However,the results of this experiment do not correspond to the literature report,and still need to be explored by subsequent further experiments. |
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