The Effect Of RhoA/ROCK Signaling Pathway On Penicillin Or Shuanghuanglian Injection Non-allergic Hypersensitivity Reactions

ObjectiveThe objective of this study was to investigate the characteristic of penicillin and Shuanghuanglian injection(SHLI)hypersensitivity reactions,to evaluate the mechanism of vascular endothelial hyperpermeability caused by these two injections,and to illuminate the effect of RhoA/ROCK signaling pathway on penicillin and SHLI induced non-allergic hypersensitivity reactions(NAHRs).In addition,the compositions involved in SHLI NAHRs were also explored in this study.Method1.Characteristic of penicillin hypersensitivity reactions(1)ICR mice were immunized by intraperitoneal injection(ip)of penicillin at 62.5,250 or 1000 kU·kg-1(aluminum hydroxide gel served as adjuvant)once every other day for three times.After 14 days,sensitized mice or naive mice were treated with a mixture consisting penicillin and evans blue(penicillin/EB)once at 31.25,125 or 500 kU·kg-1 by intravenous injection(iv).Thirty minutes after penicillin/EB administration,the score of blue area in the ears and EB extraction of the ears were evaluated to analyze the possibility of allergic hypersensitivity reactions(AHRs)and NAHRs.(2)ICR mice were immunized by penicillin as described above.After 14 days,sera were collected.The sera were injected into the dorsal skin of depilated recipient mice.After 2 hours,the recipient mice were challenged by once administration(iv)of penicillin/EB at 31.25,125 or 500 kU·kg-1.The blue dorsal regions were observed after 30 minutes.(3)ICR mice were treated(iv)with penicillin at 125,250,or 500 kU·kg-1 and euthanized 30 minutes after dosing.Punch biopsies of the ears were weighed.Ears and lungs were preserved for histopathological evaluation.2.Characteristic of SHLI hypersensitivity reactions(1)ICR mice were immunized(ip)by 300 mg·kg-1 SHLI(aluminum hydroxide gel served as adjuvant)once every other day for three times.After 14 days,sensitized mice or naive mice were treated(iv)with a mixture consisting SHLI and EB(SHLI/EB)once at 600 mg·kg-1.Thirty minutes after SHLI/EB administration,the score of blue area in the ears and EB extraction of the ears were evaluated to analyze the possibility of AHRs and NAHRs.(2)ICR mice were immunized by SHLI as described above.After 14 days,sera were collected.The sera were injected into the dorsal skin of depilated recipient mice.After 2 hours,the recipient mice were challenged by once administration(iv)of 600 mg·kg-1 SHLI/EB.The blue dorsal regions were observed 30 minutes after the challenge injection.(3)ICR mice were treated(iv)with SHLI/EB at 150,300,or 600 mg·kg-1.Thirty minutes after SHLI/EB administration,the score of blue area in the ears and EB extraction of the ears were evaluated,and punch biopsies of the ears were weighed.Ears,lungs and intestines were preserved for histopathological evaluation.3.The mechanism of penicillin NAHRs(1)The effect on human umbilical vein endothelial cells(HUVECs)monolayer:Cells were plated in a transwell insert chamber and formed endothelial monolayer.Monolayer was incubated with penicillin(1.25,5,or 20 kU·mL-1)for 1 hour,and fluorescence apparent permeability coefficients(Papp)of FITC was calculated by evaluating the diffusion of FITC-dextran through the endothelial monolayer.(2)The effect on cytoskeleton:HUVECs were cultured in a plate and incubated with penicillin(1.25,5,or 20 kU·mL-1)for 1 hour.The F-actin was stained with rhodamine-phalloidin and the alterations in cytoskeleton were observed.(3)The effect on RhoA/ROCK signaling pathway:HUVECs were cultured in a dish and incubated with 5 kU·mL-1 penicillin for 5,15,30 or 60 minutes.Then cells were collected and the expression levels of GTP-RhoA(GTP-RhoA/RhoA%),p-MYPT1(p-MYPT1/MYPT1%)and p-MLC2(p-MLC2/MLC2%)were tested by western blot assays.In addition,mice were treated(iv)with 500 kU·kg-1 penicillin,and the ears and lungs were collected at 15,30,60 or 120 minutes after dosing.The expressions of GTP-RhoA,p-MYPT1 and p-MLC2 were evaluated by western blot assays.(4)The effect of fasudil on penicillin-induced changes:HUVECs and endothelial monolayer were pretreated with fasudil hydrochloride and then incubated with penicillin.The expressions of p-MYPT1 and p-MLC2,HUVECs cytoskeleton,and permeability of endothelial monolayer were tested as described above.In addition,mice were pretreated(ip)with fasudil for three times and then received(iv)500 kU·kg-1 penicillin once.Ears and lungs were collected at 15 and 30 minutes after penicillin treatment to analyze the expressions of p-MYPT1 and p-MLC2.Mice were pretreated(ip)with fasudil for three times and then received(iv)500 kU·kg-1 penicillin/EB once.Thirty minutes after penicillin/EB administration,the score of blue area in the ears and EB extraction of the ears were evaluated,and punch biopsies of the ears were weighed.Ears and lungs were preserved for histopathological evaluation.4.The mechanism of SHLI NAHRs(1)The effect on HUVECs monolayer:HUVECs were plated in a transwell insert chamber and formed endothelial monolayer.Monolayer was incubated with SHLI(0.05,0.1 or 0.15 mg·mL-1)for 1 hour,and Papp of FITC was calculated by evaluating the diffusion of FITC-dextran through the endothelial monolayer.(2)The effect on cytoskeleton:HUVECs were cultured in a plate and incubated with SHLI(0.05,0.1 or 0.15 mg·mL-1)for 1 hour.The F-actin was stained with rhodamine-phalloidin and the alterations in cytoskeleton were observed.(3)The effect on RhoA/ROCK signaling pathway:HUVECs were cultured in a dish and incubated with 0.15 mg·mL-1 SHLI for 15,30,60 or 120 minutes.Then cells were collected and the expression levels of GTP-RhoA,p-MYPT1 and p-MLC2 were tested by western blot assays.In addition,mice were treated(iv)with 600 mg·kg-1 SHLI,and the ears,lungs and intestines were collected at 15,30,60,or 120 minutes after dosing.The expressions of GTP-RhoA,p-MYPT1 and p-MLC2 were evaluated by western blot assays.(4)The effect of fasudil on SHLI-induced changes:HUVECs and endothelial monolayer were pretreated with fasudil hydrochloride and then incubated with SHLI.The expressions of p-MYPT1 and p-MLC2,HUVECs cytoskeleton,and permeability of endothelial monolayer were tested as described above.In addition,mice were pretreated(ip)with fasudil for three times and then received(iv)600 mg·kg-1 SHLI once.Ears,lungs and intestines were collected at 30 minutes after SHLI treatment to analyze the expressions of p-MYPT1 and p-MLC2.Mice were pretreated(ip)with fasudil for three times and then received(iv)600 mg·kg-1 SHLI/EB once.Thirty minutes after SHLI/EB administration,the score of blue area in the ears and EB extraction of the ears were evaluated,and punch biopsies of the ears were weighed.Ears,lungs and intestines were preserved for histopathological evaluation.5.Forsythiaside NAHRs and the mechanism(1)Research on NAHRs:HUVECs were plated in a transwell insert chamber and formed endothelial monolayer.Monolayer was incubated with forsythiaside A,B or E(0.05,0.1 or 0.15 mg·mL-1)for 1 hour,and Papp of FITC was calculated by evaluating the diffusion of FITC-dextran through the endothelial monolayer.HUVECs were cultured in a plate and incubated with forsythiaside A,B or E(0.05,0.1 or 0.15 mg·mL-1)for 1 hour.The F-actin was stained with rhodamine-phalloidin and the alterations in cytoskeleton were observed.Mice were treated with(iv)600 mg·kg-1 forsythiaside A,B or E/EB once.Thirty minutes after drug/EB administration,the score of blue area in the ears and EB extraction of the ears were evaluated.(2)Research on the mechanism:HUVECs were cultured in a dish and incubated with 0.05,0.1 or 0.15 mg·mL-1 forsythiaside A,B or E.Then cells were collected and the expression levels of GTP-RhoA,p-MYPT1 and p-MLC2 were tested by western blot assays.HUVECs and endothelial monolayer were pretreated with fasudil hydrochloride and then incubated with forsythiaside A or B.The expressions of p-MYPT1 and p-MLC2,HUVECs cytoskeleton,and permeability of endothelial monolayer were evaluated as described above.Result1.Penicillin could induce mice NAHRsWhen treated(iv)with penicillin/EB,sensitized mice and unsensitized mice exhibited similar EB extravasation.This reaction was dose-dependent,and not related with sensitization.The PCA test result manifested that IgE was not elicited by penicillin,which indicated that IgE was not responsible for the penicillin-induced reaction.This reaction might belong to NAHRs.The microscopic examination confirmed that treatment(iv)with penicillin once could cause dose-dependent edema and inflammation in the ears and lungs of mice.These results demonstrate that penicillin could induce mice NAHRs,which are characterized by increased vascular leakage and inflammation.2.SHLI could induce mice NAHRsWhen treated(iv)with SHLI/EB,sensitized mice and unsensitized mice could exhibit similar EB extravasation.This reaction was not related with sensitization.The PCA test result manifested that IgE was not elicited by SHLI,which indicated that IgE was not responsible for the SHLI-induced reaction.This reaction might belong to NAHRs.In addition,SHLI-induced EB extravasation was dose-dependent.The microscopic examination confirmed that treatment(iv)with SHLI once could cause edema and inflammation in the ears,lungs and intestines of mice.These results demonstrate that SHLI could induce mice NAHRs,which are characterized by increased vascular leakage and inflammation.3.RhoA/ROCK signaling pathway contributes to penicillin-induced NAHRsIn the in vitro experiments,incubation with 5 or 20 kU·mL-1 penicillin could result in noticeable F-actin accumulation and reorganization in HUVECs,and significantly enhance the permeability of endothelial monolayer.Both changes were concentration-related.Incubation with 5 kU·mL-1 penicillin also upregulated the expressions of GTP-RhoA,p-MYPT1 and p-MLC2 in HUVECs.The protein levels were significantly increased within 15-30 minutes of penicillin exposure and then gradually declined.Pretreatment with fasudil could alleviate penicillin-induced endothelial hyperpermeability and alterations in cytoskeleton.In the in vivo experiments,when mice were treated(iv)with 500 kU·kg-1 penicillin,protein levels of GTP-RhoA,p-MYPT1 and p-MLC2 in the ears and lungs were augmented.These proteins were expressed at peak levels within 15-30 minutes of penicillin exposure and then gradually declined.Pretreatment with fasudil significantly attenuated penicillin-induced increased vascular leakage and inflammation.These results show that RhoA/ROCK signaling pathway contributes to penicillin NAHRs.4.RhoA/ROCK signaling pathway contributes to SHLI-induced NAHRsIn the in vitro experiments,incubation with 0.1 or 0.15 mg·mL-1 SHLI could result in F-actin accumulation and reorganization in HUVECs,and enhance the permeability of endothelial monolayer.Both changes were concentration-related.Incubation with 0.15 mg·mL-1 SHLI also upregulated the expressions of GTP-RhoA,p-MYPT1 and p-MLC2 in HUVECs.The protein levels were significantly increased within 15-30 minutes of SHLI exposure and then gradually declined.Pretreatment with fasudil could alleviate SHLI-induced endothelial hyperpermeability and alterations in cytoskeleton.In the in vivo experiments,when mice were treated(iv)with 600 mg·kg-1 SHLI,protein levels of GTP-RhoA,p-MYPT1 and p-MLC2 in the ears,lungs and intestines were augmented.These proteins were expressed at peak levels within 15-60 minutes of SHLI exposure and then gradually declined.Pretreatment with fasudil significantly attenuated SHLI-induced increased vascular leakage and inflammation.These results show that RhoA/ROCK signaling pathway contributes to SHLI NAHRs.5.RhoA/ROCK signaling pathway contributes to forsythiaside-induced NAHRs In the in vitro experiments,incubation with 0.1 or 0.15 mg·mL-1 forsythiaside A or B could enhance the permeability of endothelial monolayer.However,forsythiaside E had little impact on endothelial permeability.In accordance with the result of permeability experiment,0.1 or 0.15 mg·mL-1 forsythiaside A or B could lead to alterations in cytoskeleton,which were almost impossible to observe in forsythiasideE-treated cells.Treatment with forsythiaside A or B caused concentration-related upregulation of GTP-RhoA,p-MYPT1 and p-MLC2,in which forsythiaside A showed stronger effect than forsythiaside B.Forsythiaside E had little effect on the expressions of these proteins.Pretreatment with fasudil could inhibit endothelial hyperpermeability and alterations in cytoskeleton elicited by forsythiaside A or B.These results were confirmed in the in vivo experiment.Forsythiaside A or B could lead to increased vascular leakage in mice.However,no reaction was observed in forsythiaside E-treated animals.These results indicate that RhoA/ROCK signaling pathway plays an important role in forsythiaside-induced NAHRs,and the effects are correlated with the structures of the compositions.Conclusion1.Both penicillin and SHLI could cause mice NAHRs,which are characterized by increased vascular leakage and inflammation.IgE is not elicited by these two injections.The reactions induced by these two injections are both in a dose-dependent manner.These results indicate that in clinical practice,when the dosage of penicillin and SHLI are controlled,it is possible to attenuate the adverse reactions induced by these two injections.2.The activation of RhoA/ROCK signaling pathway which could cause alterations in cytoskeleton and increased vascular leakage plays an important role in penicillin or SHLI NAHRs.3.Forsythiaside is one of the main compositions which involve in SHLI NAHRs.The effect is correlated with the structure of forsythiaside.Forsythiaside A has stronger impact than forsythiaside B,and forsythiaside E shows no effect.RhoA/ROCK signaling pathway also contributes to forsythiaside-induced NAHRs.