| Objective:In recent years,the incidence of invasive fungal infection(IFIs),with Candida albicans(CA)as the main pathogen,has increased which poses a great threat to human health.Fluconazole(FLC)as a clinical first-line drug,the drug resistance rate increases gradually with its widespread use,which brings difficulties for clinical treatment.Traditional Chinese medicine has become a research hotspot in new drug development because of its many targets and isn’t easy to produce drug resistance,and has the advantages of low toxicity effects,wide range of sources,low price and so on.High permeability glycerol mitogen activated protein kinase signal pathway(HOG-MAPK)is the most representative pathway for Candida albicans to adapt to external pressure.This pathway regulates the adaptation of Candida albicans to external osmotic and oxidative pressure,cell morphological transformation and cell wall synthesis,and can be used as a potential target for drug intervention.In the previous study,we found that Berberine hydrochloride(BBH),the main active component of Coptis,had good inhibitory effect on planktonic and biofilm Candida albicans.In this study,BBH was selected as the intervention drug to explore the inhibitory effect and molecular mechanism of berberine hydrochloride on Candida albicans by analyzing the effects of BBH on the five functions of HOG-MAPK pathway and the expression of key genes and proteins in Candida albicans,so as to provide new ideas and methods for clinical treatment of drug-resistant Candida albicans infection.Methods:1.The minimum inhibitory concentration(MIC)of berberine hydrochloride and fluconazole in inhibiting the growth of Candida albicans was determined by the microdilution method.The intervention concentration of berberine hydrochloride in this study was used as 4MIC,MIC,1/4MIC while the intervention concentration of fluconazole was 4MIC.2.The intracellular glycerol content of Candida albicans was measured by Trinder method,the level of reactive oxygen species(ROS)was determined by DCFH-DA fluorescence probe,the content of ATP was determined by chemiluminescence,and the levels of chitin and β-1,3-glucan of cell wall in Candida albicans cells were measured by fluorescent dye method..The intracellular ROS level,hypha formation and germ tube formation rate of Candida albicans were observed by inverted fluorescence microscope,and the exposure of cell wall chitin was observed by laser confocal microscope(CLSM).To study the effects of berberine hydrochloride on five functions of HOG-MAPK pathway in Candida albicans.3.Real-time fluorescence quantitative PCR(RT-q PCR)was used to detect the expression of key genes(HOG1,PBS2,CHS3,GSC1)of HOG-MAPK central signaling pathway after the intervention of berberine hydrochloride.The expression level of HOG1 protein in Candida albicans was detected by Western Blot to discusse the molecular regulation mechanism of berberine hydrochloride on HOG-MAPK central signaling pathway in Candida albicans.Results:1.The MIC of berberine hydrochloride of 14 fluconazole resistance Candida albicans clinical strains and standard strain ATCC10231 was 64 μg/m L while the MIC of fluconazole against ATCC10231 was 1 μg/m L.According to the MIC value,the subsequent intervention concentrations of berberine hydrochloride were 256 μg/m L,64 μg/m L and 16 μg/m L respectively,and the intervention concentrations of positive control fluconazole was 4 μg/m L.2.For intracellular osmotic pressure: 256 μg/m L and 64 μg/m L berberine hydrochloride could significantly increase the intracellular glycerol content and cell osmotic pressure of ATCC10231 and CA clinical strains,and the effect of 256 μg/m L berberine hydrochloride was better than that of 64 μg/m L berberine hydrochloride and 4 μg/m L fluconazole(P<0.05),but 16μg/m L berberine hydrochloride could only increase the intracellular glycerol content of ATCC10231(P<0.05),but had no significantly effect on CA clinical strains(P>0.05).At the same time,the intracellular glycerol content in ATCC10231 treated with 256 μg/m L and 16 μg/m L berberine hydrochloride was higher than that of CA clinical strains(P<0.05).3.For intracellular ROS level: 256 μg/m L and 64 μg/m L berberine hydrochloride could significantly increase the intracellular ROS level of ATCC10231 and CA clinical strains,and the effect of 256 μg/m L berberine hydrochloride was better than that of 64 μg/m L berberine hydrochloride but less than 4 μg/m L fluconazole(P<0.05).16 μg/m L berberine hydrochloride could only increase the intracellular ROS level of ATCC10231(P<0.05),but had no significantly effect on CA clinical strains(P>0.05).At the same time,the intracellular ROS level of CA clinical strain was higher than that of ATCC10231 in 256 μg/m L and 64 μg/m L berberine hydrochloride groups(P<0.05).The intracellular fluorescence intensity observed by inverted fluorescence microscope was consistent with that of DCFH-DA fluorescence probe method.4.For intracellular ATP content: 256 μg/m L and 64 μg/m L berberine hydrochloride could significantly increase the intracellular ATP content of ATCC10231 and CA clinical strains,and the effect of 256 μg/m L berberine hydrochloride was better than that of 64 μg/m L berberine hydrochloride but less than 4 μg/m L fluconazole(P<0.05).16 μg/m L berberine hydrochloride could only increase the intracellular ATP content of ATCC10231(P<0.05),but had no significantly effect on CA clinical strains(P>0.05).At the same time,the intracellular ATP content in ATCC10231 treated with 256 μg/m L and 64 μg/m L berberine hydrochloride was higher than that of CA clinical strains(P<0.05).5.For cell morphological transformation: 256 μg/m L and 64 μg/m L berberine hydrochloride could significantly inhibit germ tube formation of ATCC10231 and CA clinical strains(P<0.05).16 μg/m L berberine hydrochloride could inhibit germ tube formation only at 4 hs(P<0.05),but not at 6 hs(P>0.05).At the same time,256 μg/m L and 64 μg/m L berberine hydrochloride had better inhibitory effect on germ tube formation of ATCC10231 than CA clinical strain(P<0.05).256 μg/m L and 64 μg/m L berberine hydrochloride could significantly inhibit the hypha formation of ATCC10231 and CA clinical strains,and the inhibitory effect of 256 μg/m L berberine hydrochloride was better than that of 64 μg/m L berberine hydrochloride and 4 μg/m L fluconazole,while 16 μg/m L berberine hydrochloride had no obviously inhibitory effect..6.For cell wall chitin exposure: CLSM showed that the cell wall fluorescence intensity of 256 μg/m L and 64 μg/m L berberine hydrochloride groups of ATCC10231 and CA clinical strains was significantly enhanced,and the fluorescence intensity of 256 μg/m L berberine hydrochloride group was higher than that of 4 μg/m L fluconazole(P<0.05).16 μg/m L berberine hydrochloride had no significantly effect on cell wall chitin(P>0.05).256 μg/m L and 64 μg/m L berberine hydrochloride could significantly increase the chitin level of ATCC10231 and CA clinical strains,and the effect of 256 μg/m L berberine hydrochloride was better than that of 64 μg/m L berberine hydrochloride and 4 μg/m L fluconazole(P<0.05).16μg/m L berberine hydrochloride had no significantly effect on cell wall chitin(P>0.05).At the same time,the chitin level of ATCC10231 was higher than that of CA clinical strains in 256 μg/m L and 64 μg/m L berberine hydrochloride groups(P<0.05).7.For the exposure of cell wall β-1,3-glucan: 256 μg/m L and 64 μg/m L berberine hydrochloride could significantly increase the cell wall β-1,3-glucan of ATCC10231 and CA clinical strains,and the effect was better than that of 4 μg/m L fluconazole(P<0.05).16 μg/m L berberine hydrochloride could only increase the cell wall β-1,3-glucan of ATCC10231(P<0.05),but had no significantly effect on CA clinical strains(P>0.05).At the same time,the level of β-1,3-glucan in ATCC10231 cell wall of 64 μg/m L berberine hydrochloride group was higher than that of CA clinical strain,while 256 μg/m L berberine hydrochloride group ATCC10231 cell wall β-1,3-glucan level was lower than that of CA clinical strain(P<0.05).8.Effect on the expression of key genes of HOG-MAPK central signaling pathway: 256 μg/m L and 64 μg/m L berberine hydrochloride could significantly up-regulate the expression of HOG1 and PBS2 of ATCC10231 and CA clinical strains,and down-regulate the expression of GSC1,and the effect was better than that of 4 μg/m L fluconazole(P<0.05).256μg/m L berberine hydrochloride could significantly down-regulate the expression of CHS3 in clinical strains ATCC10231 and CA clinical strains(P<0.05),and 64 μg/m L berberine hydrochloride could only down-regulate the expression of CHS3 in clinical strains of CA(P<0.05),but had no significantly effect on ATCC10231(P>0.05).While 16 μg/m L berberine hydrochloride had no significantly effect on the expression of these related genes(P>0.05).9.Effect on the expression of key protein of HOG-MAPK pathway: 256 μg/m L berberine hydrochloride could significantly up-regulate the expression of HOG1 protein in ATCC10231 and CA clinical strains(P<0.05),and there was no significantly difference compared with 4 μg/m L fluconazole(P>0.05),while 64 μg/m L and 16 μg/m L berberine hydrochloride had no significantly effect on HOG1 protein expression(P>0.05).Conclusion:1.Berberine hydrochloride can inhibit Candida albicans by affecting five functions of HOG-MAPK pathway,including increasing intracellular glycerol content to increase osmotic pressure,increasing ROS production to promote cell oxidative stress and killing,promoting mitochondrial respiratory metabolism to promote ROS production,inhibiting germ tube and hypha formation to hinder morphological transformation,increasing cell wall chitin and β-1,3-glucan exposure to destroy cell wall integrity.2.The molecular mechanism of berberine hydrochloride on the HOG-MAPK pathway of Candida albicans may be related to the up-regulation of the core genes of the central signal pathway,HOG1 and PBS2,down-regulation the expression of effector genes CHS3 and GSC1,and increasing the accumulation of HOG1 protein in cells.However,the exact mechanism needs to be further studied.3.On the whole,the effect of berberine hydrochloride on the function of HOG-MAPK pathway of drug resistant clinical strains is not as good as that of standard strains.It suggested that the production of fluconazole resistance may increase the resistance of fungi to other drugs,resulting in a decrease in fungicidal effect,and the specific reasons need to be further studied by increasing the sample accounts. |
