Discovery And Molecular Pharmacology Of Chloride Channel Inhibitors In Xingnaojing

Xingnaojing is a traditional Chinese medicine injection with the functions of awakening the brain,cooling blood,promoting blood circulation,removing blood stasis,clearing heat and detoxifying.It is often used to treat consciousness disorders caused by cerebral hemorrhage,cerebral infarction,craniocerebral trauma,stroke,central nervous system infection and other acute cerebrovascular diseases.Xingnaojing can make the brain sharp and quickly cause consciousness,and is widely used in the acute phase of cerebral hemorrhage(ICH).Although Xingnaojing has a definite curative effect and is widely used in clinical practice,the clinical safety of Xingnaojing injection has been widely valued because its dosage form is an injection form and it shows various side effects in clinical application.Nebulized inhaler is a relatively safe new form of administration.The way of medicine can greatly reduce the side effects of injection administration.The maintenance of respiratory tract function is inseparable from the appropriate amount of water and mucus secretion.Therefore,the ideal aerosol preparation should not affect the clearance function of airway mucus.In order to determine the feasibility of inhalation preparation of the development of Xingnaojing into aerosols,Xingnaojing and its active components were used to analyze the effects of Xingnaojing and its active components on various airway fluid secretion-related chloride channels using cell fluorescence functional model,short-circuit current technology,and high-performance liquid HPLC technology.The experimental results are as follows:1.Using the FRT/TMEM16A/YFP-H148Q/I152L functional assay model,it shows that Xingnaojing can inhibit TMEM16A channel activity in a dose-dependent manner,with an IC50 value of about 23.37 μg/mL.2.The results of short-circuit current experiments on FRT-TMEM16A cells showed that Xingnaojing can inhibit TMEM16A-mediated currents on the apical side of FRT-TMEM16A cells,with an IC50 value of about 19.65 μg/mL.3.The FRT/CFTR/YFP-H148Q/I152L functional assay screening model detected that Xingnaojing can inhibit CFTR channel activity in a dose-dependent manner,with an IC50 value of approximately 40.06 μg/mL.4.The results of short-circuit current experiments on FRT-CFTR cells show that Xingnaojing can inhibit the CFTR-mediated current on the apical side of FRT-CFTR cells,and achieve 69.56%inhibition rate at 134 μg/mL.5.HPLC analysis results showed that the concentrations of curzerenone,curdione,and gemacrone in Xingnaojing were 1.09 μM(curzerenone),8.23 μM(curdione),3.1 μM(gemacrone),and furanodienone and curcumol content is extremely low.6.Using the FRT/TMEM16/YFP-H148Q/I152L functional assay model,it has been detected that curzerenone,germacrone,furanodienone,curcumol and curdione can inhibit TMEM16A channel activity in a dose-dependent manner,with IC50 values about 87.96 μM(curzerenone),15.12 μM(germacrone),23.35 μM(furanodienone),50.55 μM(curcumol),44.00 μM(curdione).7.The analysis results of short-circuit current experiments on FRT-TMEM16A cells showed that sesquiterpenoids can inhibit TMEM16A-mediated current on the apical side of FRT-TMEM16A cells.The IC50 values are about 16.32 μM(curzerenone),11.45 μM(germacrone),9.88 μM(furanodienone),30.58 μM(curcumol)and 38.62 μM(curdione).The five sesquiterpenes have a rapid inhibitory effect on TMEM16A calcium-activated chloride channel activity.Among them,curdione,curcumol and furanodienone have irreversible inhibitory effects,and curzerenone and germacrone have reversible inhibitory effects.8.The results of the measurement of the effect of CFTR show that sesquiterpenoids have an inhibitory effect on CFTR channel activity.Among them,curzerenone and germacrone can completely inhibit the chloride ion current at 20 μM.Curcumol can completely inhibit the chloride ion current at 200 μM.Curdione and furanodienone at 83.78%and 91.67%respectively at 20 μM.The results of short-circuit current on the tissues showed that sesquiterpenoids had no inhibitory effect on the CFTR chloride channel.9.The short-circuit current measurement results on HBE cells showed that the five sesquiterpenoids could inhibit the current generated by UTP,and the inhibition rates were 65.12%(curzerenone),91.16%(germacrone),and 79.07%(furanodienone),76.74%(curcumol),67.44%(curdione).Among them,the inhibitory effect of germacrone is the most obvious,which is similar to the positive control CaCCinh-A01(90.69%).10.The results of Ca2+ concentration measurement show that sesquiterpenes can inhibit the increase of intracellular calcium ion concentration caused by ATP except curdione,indicating that curzerenone,germacrone,furanodienone and curcumol can inhibit CaCC chloride channel activity by within the calcium ion concentration.11.The results of the measurement of the effect of CaCC show that all five compounds can inhibit the ATP-induced CaCC current,and the maximum inhibitory concentrations are 50 μM(curzerenone),50 μM(germacrone),50 μM(furanodienone),100 μM(curcumol),200μM(curdione).12.After incubation,the five sesquiterpenoids on the tissue have an inhibitory effect on the current induced by cch.Curzerenone inhibited the currents by 48.21%and 67.89%at 50μM and 100 μM,respectively.Germacrone inhibited currents of 26.65%and 41.44%at 50 μM and 100 μM.Furanodienone of 50 μM and 100 μM inhibited currents of 14.24%and 34.89%.Curcumol of 100 μM and 200 μM inhibited 22.40%and 33.78%of the current.Curdione of 100 μM and 200 μM inhibited 11.65%and 30.72%of the current,and the curative effect of curzerenone 50 μM and 100 μM is better than that of CaCCinh-A01.13.The measurement results of Na+-K+-ATPase channel showed that five sesquiterpenoids had no inhibitory effect on Na+-K+-ATPase on the serous side of mouse colon epithelium.14.The measurement results of the K+channel showed that the five sesquiterpenoids inhibited the activity of the K+channel at 100 μM,and the inhibition rates were 72.66%(curzerenone),10.18%(germacrone),19.69%(furanodienone),41.58%(curcumol)and 20.27%(curdione),of which curzerenone has the most significant inhibitory effect.In summary,Xingnaojing and its active ingredients have no inhibitory effect on the activity of TMEM16A,CFTR and CaCC chloride channels under low concentration conditions,suggesting that there is no significant effect on the transport of water and mucus,and the nebulization administration scheme is feasible.High concentration of Xingnaojing can inhibit the activity of TMEM16A and CFTR chloride channel.And at high concentration,the active compound can reduce intracellular Ca2+ concentration and inhibit potassium channel activity.So pay attention to the use of this drug in large doses,or long-term administration.The resulting drug accumulation can still cause side effects.