| BackgroundWith the elevated incidence of lung cancer in recent decades,there is a gradual expansion in the application of targeted drugs in clinical practice.While corresponding adverse reactions（e.g.,dermatitis and rash）are also becoming increasingly common clinically.At present,the issue is that it is still unclear with respect to the specific cause and pathogenesis of skin toxicity caused by targeted drugs such as erlotinib.In this regard,there are no obvious effective and targeted drugs and therapeutic approaches.According to traditional Chinese medicine,this disease belongs to the categories of"drug toxicity","drug toxicity rash",and"furuncle and prickly heat".Its pathogenesis can be explained by the internal endowment weakness,insecurity of the grain of the skin and the texture of the subcutaneous flesh,and external invasion of drug toxicity,resulting in the toxic wind,heat and dampness stagnating in the skin,the grain of the skin and the texture of the subcutaneous flesh firstly.Consequently,it induces a blood heat-induced rash syndrome dominated by red macula,or a damp heat-induced rash syndrome dominated by red spots and blisters.Its general pathogenesis includes internal endowment weakness and toxin invasion.At present,there is no unified treatment standard of traditional Chinese and Western medicine for the treatment of dermatitis and rash caused by targeted drugs.Currently,treatment using Western medicine shows certain limitations since its treatment is based on hormone and antibiotic ointment,such as Dexamethasone Acetate Ointment,Hydrocortisone Ointment,Erythromycin Ointment,Clindamycin Gel,urea ointment,oral minocycline,etc.The existing research of modern traditional Chinese medicine has got some achievements,and has made some exploration on the traditional Chinese medicine classification,clinical efficacy,internal and external treatment of dermatitis.Nevertheless,the disadvantage lies in the limited sample size during clinical observation,resulting in the absence of evidence-based medical data.In this experiment,on the basis of relevant domestic and foreign literatures,an animal model of TKI-induced dermal inflammatory reaction was established and verified by macroscopic observation of the changes of the skin surface,pathological sections,inflammatory cells and cytokines in mice before and after treatment with erlotinib.Simultaneously,this study further explored the pathogenesis of erlotinib-related dermatitis to provide an animal model for evaluating the effectiveness and mechanism of drugs or acupuncture.Zusanli is the main acupoint of"Yangming Stomach Meridian of Foot",which is located on the anterolateral side of the lower leg,3 inches below the Dubiacupoint,and a horizontal finger（middle finger）outside the front edge of the tibia.It is the confluent point of Yangming Stomach Meridian of Foot and the lower confluent point of the stomach,one of the four command points.Zusanli has been recognized to be an important acupoint for health care.According to traditional Chinese medicine,massaging Zusanli can regulate immunity,enhance the ability of disease resistance,regulate the spleen and stomach,tonify middle-Jiao and Qi,dredge meridians and activate collaterals,dispel toxic wind and dampness,and strengthen the body resistance and eliminate pathogenic factors.In the field of traditional Chinese medicine,Zusanli acupoint is favored by doctors of all dynasties owing to its special function and wide application.Acting as the confluent acupoint of Yangming Stomach Meridian of Foot,it plays a critical role in regulating QI and blood to exert the function of reinforcing weakened body and building up health,which has a therapeutic effect covering various diseases.It can be considered that,the scope of indications of Zusanli acupoint ranges from head to foot.Zusanli has theeffect of building up a good physique and improving one’s health.It is one of the important acupoints for supporting healthy energy and preventing diseases.It also has an intimate association with the viewpoint of preventive medicine to eliminate the"transmission and transformation of pathogenic factors".It has been recorded in"Neijing（Inner Canon of the Yellow Emperor）"that"A good doctor generally follows the therapeutic principle of preventive treatment of disease,rather than treatment of the already developed disease.What’s the point of knowing to take medicine for treatment after the onset of disease?"Zhang Zhongjing also emphasized in the"Synopsis of Prescriptions of the Golden Chamber"of the Han Dynasty that"In the preventive treatment of disease,when there is a disease of the liver,it shall be noted that it usually originates from the spleen and hence the therapeutic principle should be reinforcing the spleen".Collectively,these discussions all emphasize the importance of preventive treatment of diseases,which strongly reveals the view of prevention-oriented prevention and control.There are multiple modern studies on acupuncture and moxibustion at Zusanli and other acupoints to enhance human immune function.Regular acupuncture and moxibustionat these acupoints can invigorate blood circulation,regulate the function of metabolism,and enhance resistance.Moreover,it can improve the immune function of the body from many aspects and enhance the immune defense ability.In traditional Chinese medicine,Zusanli acupoint is usually considered to regulate QI and blood,so as to eliminate fatigue and increase appetite.Corresponding indications include diseases of nose,mouth,face,forehead,breast,diaphragm,stomach,spleen,large intestine,small intestine and the parts with the distribution of meridians.Furthermore,with the development of immunology,it is found that Zusanli acupoint has a relationship with immunity,which is manifested in enhancing the phagocytosis of leukocytes,associating with the formation of antibodies,and having a certain impact on lectin,hemolysin and bactericide,etc.Clearly,acupuncture at Zusanli can replenish QI and blood,strengthen the body,stimulate and mobilize the internal systemic acquired resistance of the human body,mobilize the deficiency and excess state of the body,so as to achieve the purpose of controlling the transmission and transformation of pathogenic factors and curing diseases.In this way,there will be a low risk of disease attack in the presence of abundant stomach QI,sufficient Yang QI,vital essence and blood.Exactly owing to the prominent effects of Zusanli acupoint in tonifying and strengthening the body resistance to promote or enhance various specific and non-specific immune functions in vivo,which is commonly used in acupuncture and moxibustion in the treatment of dermal inflammatory reaction.Past research supports that electroacupuncture（EA）has anti-allergic and anti-inflammatory effects.Accordingly,the present study was carried out to explore the regulatory mechanism of EA pretreatment at Zusanli on TKI-induced dermal inflammatory reaction by observing the inhibitory effect of EA on erlotinib-induced dermal inflammatory reaction in mice.Meanwhile,our study also discussed whether EA pretreatment at Zusanli would have an impact on the efficacy of erlotinib.ObjectiveBased on the erlotinib-induced dermal inflammatory reaction model in BALB/C mice,successful modeling was determined from the aspects of the changes of epidermis,dermis,epidermis+dermis before and after EA intervention via HE staining of the skin,the expression of KRT14 protein in skin keratinocytes and the expression of LY6G in skin tissue.At the same time,further experiments were performed to observe the changes of TRPV1 and IL-33 in the skin tissue of different groups of mice,and to explore the inhibitory effect of EA pretreatment at Zusanli on the dermal inflammatory reaction by altering the local proinflammatory factor IL-33 through downregulating the expression of TRPV1.Methods1.Female BALB/C mice of clean grade（n=40）were selected and raised for 7 days routinely,which were then randomly divided into 4 groups,with 10mice in each group.The specific grouping and processing were as follows:the normal control group（group 1,normal BALB/C mice）,the model group（group2,erlotinib-induced model group）,EA pretreatment+model group（group 3,EA+erlotinib-induced model group,modeling after 7 days of EA pretreatment at Zusanli bilaterally）,and EA pretreatment alone group（group 4,with 7 days of EA pretreatment at Zusanli bilaterally）.Except for the normal control group,mice in the other groups were orally perfused with erlotinib solution at a dose of l00mg/kg（Erlotinib Hydrochloride Tablets dissolved in sterilized deionized water and prepared into l0g/L erlotinib solution）,once a day.Mice in the normal control group were gavaged with an equal volume of sterilized deionized water.The gavage time was set to be 5 days since the skin of BALB/C mice showed an obvious dermal inflammatory reaction on the 5th day after gavage that could be observed under the microscope.The time of acupuncture treatment was 9:00-12:00 every morning,and that of intragastric administration was controlled between 16:00-18:00 every afternoon.2.EA pretreatment:The stimulation intensity of EA was set at the minimum voltage that could cause moderate muscle contraction,and the parameters were as follows:continuous wave at 2Hz for 5 min（0.3m A）;continuous wave at 2Hz for 5 min（0.5m A）;and continuous wave at 2Hz for20min（1m A）.The continuous EA pretreatment was performed for 7 days（started at 9:00 a.m,30min each time,once a day）.3.After EA pretreatment+modeling,on the 13th day,the mice were anesthetized by intraperitoneal injection of 5%chloral hydrate.With the hair of mice shaved,the chest skin was taken,fixed and embedded,and made into sections for HE staining and immunohistochemical detection.Skin homogenate and serum were detected for immunoglobulin and cytokines by ELISA.The related proteins in the spleen and skin were detected by the Western blotting method.Experiment Ⅰ1.Thickness changes of epidermis,dermis,epidermis+dermis of mice in each group before and after EA intervention were observed via HE staining under the routine light microscope,which were measured at the same time.2.KRT14 and LY6G proteinexpressions were detected via immunohistochemistry in the skin of mice in each group before and after EA pretreatment.3.KRT14 protein expression was evaluated by Western blotting in mouse skin before and after EA pretreatment.4.The changes of immunoglobulin antibodies（total Ig G,Ig G1,Ig G2a and Ig G3）in serum,inflammatory factors（IFN-γ,IL-5,IL-17,IL-1β,and IL-33）were detected via ELISA in serum and skin homogenate before and after EA pretreatment.5.The changes of TRPV1 levels were detected via Western blottingin the skin and spleen of mice before and after EA pretreatment.6.The expressions of KRT14 and TRPV1 were detected via immunofluorescence in the skin of mice in each group.Experiment Ⅱ1.The changes of TRPV1 levels were detected via Western blottingin mouse skin and spleen of the four groups（model group,EA+model group,AMG-517+EA+model group,Capsicine+EA+model group）before and after the application of TRPV1 inhibitor AMG-517 and TRPV1 agonist Capsicine.2.Thickness changes of epidermis,dermis,epidermis+dermis of mice in each group were observed via HE staining under the routine light microscope,which were measured at the same time.3.KRT14 protein expression was detected via immunohistochemistry and Western blotting in mouse skin of the four groups.4.LY6G protein expression was detected via immunohistochemistry in mouse skin of the four groups.5.The changes of IL-33 were detected via ELISA in serum and skin homogenate of mice in the four groups.Experiment Ⅲ1.Intraperitoneal injection of IL-33:（1）The general appearance of the skin（the degree of skin swelling and congestion）were observed in the four groups of mice（dd H2O intraperitoneal injection group,erlotinib-induced model group,IL-33 intraperitoneal injection+erlotinib-induced model group,IL-33 intraperitoneal injection group）,associated with macroscopic observation of the blood vessels of the four groups of mice.（2）Thickness changes of epidermis,dermis,epidermis+dermis of mice in four groups of mice were observed via HE staining under the routine light microscope,which were measured at the same time.（3）KRT14 proteinexpression was detected via immunohistochemistry and Western blottingin mouse skin of the four groups.（4）The expression of TRPV1 was detected by Western blottingin mouse skin and spleen of the four groups.2.Intradermal injection of IL-33:（1）The general appearance of the skin（the degree of skin swelling and congestion）were observed in the four groups of mice（dd H2O intradermal injection group,erlotinib-induced model group,IL-33 intradermal injection+erlotinib-induced model group,IL-33 intradermal injection group）,associated with macroscopic observation of the blood vessels of the four groups of mice.（2）Thickness changes of epidermis,dermis,epidermis+dermis of mice in four groups of mice were observed via HE staining under the routine light microscope,which were measured at the same time.（3）KRT14 protein expression was detected via immunohistochemistry and Western blottingin mouse skin of the four groups.（4）The expression of TRPV1 was detected by Western blottingin mouse skin and spleen of the four groups.Experiment Ⅳ1.The protein expression of EGFR was detected by immunohistochemistry in mouse skin tissue of the model group and EA+model group.2.The protein expression of EGFR was detected by Western blotting in mouse skin and spleen of the model group and EA+model group.Results1.EA pretreatment at Zusanli could effectively inhibit the erlotinib-induced dermal inflammatory reaction.According to the results of histomorphological observation,compared with the erlotinib-induced model group,EA pretreatment at Zusanli could reduce the thickness of skin epidermis and dermis in the model group,leading to an alleviation in the thickening of skin epidermis and dermis after EA intervention,with statistically significant difference（P<0.05）.The detection results of ELISA revealed that EA pretreatment could downregulate serum immunoglobulin（Ig G,Ig G-1,Ig G-2A,Ig G-2B and Ig-G3）expressions to varying degrees（P<0.01,P<0.0001,P<0.0001,P<0.0001,P<0.0001）,and inhibit the release of serum inflammatory factors（1L-1β,IFN-γ,IL-5,IL-17and IL-33）in serum（P<0.0001,P<0.001,P<0.001,P<0.001,P<0.05）and in skin homogenate（P<0.001,P<0.05,P<0.05,P<0.01,P<0.0001）of mice from the model group.Meanwhile,the results of immunohistochemistry indicated that after EA pretreatment at Zusanli,the protein expressions of KRT14 and LY6G in the EA+model group decreased compared with those in the model group,with statistically significant differences（P<0.05,P<0.01）.While there was no statistically significant difference in the protein expressions of KRT14and LY6G between the normal control group and the EA pretreatment alone group（P>0.05）.Based on further detection using Western blotting,there was a statistically significant difference that the protein expression of KRT14 was reduced to some extent after EA pretreatment at Zusanli than that in the model group（P<0.05）,which was consistent with that detected by immunohistochemistry.Immunofluorescence assay revealed that KRT14 and TRPV1 were significantly expressed in mouse skin.Moreover,considering the immunofluorescence intensity of KRT14 and TRPV1,corresponding expression trends among groups were consistent with those tested by immunohistochemistry and Western blotting.There was a statistically significant difference that the expressions of KRT14 and TRPV1 in mouse skin of the model group were increased when compared with the normal control group（P<0.05）.After EA pretreatment at Zusanli,the expressions of KRT14 and TRPV1 were reduced than those in the erlotinib-induced model group,with a statistically significant difference（P<0.05）.However,no statistically significant differencewas observed between the normal control group and the EA pretreatment alone group（P>0.05）.2.EA pretreatment at Zusanli could inhibit the expression of TRPV1 in mouse skin and spleen.The results of Western blotting showed that the protein expressions of TRPV1 in mouse skin and spleen were reduced to some extent after EA pretreatment at Zusanli than those in the model group,with a statistically significant difference（P<0.05）.But no statistically significant differencewas detected when compared EA pretreatment alone group to the normal control group（P>0.05）.3.EA pretreatment at Zusanli could inhibit the expression of IL-33 in mouse serum and skin homogenate,of which IL-33 could promote the dermal inflammatory reaction.The results of ELISA showed that there was a statistically significant difference that the expression of IL-33 in mouse serum and skin homogenate were reduced to some extent after EA pretreatment at Zusanli than those in the model group（P<0.01,P<0.0001）.While no statistically significant differencewas observed between the normal control group and EA pretreatment alone group（P>0.05）.Through gross and macroscopic observation（epidermis and vascular surface）,histomorphological observation based on HE staining showed that compared with the model group,there was an increase in the thickness of skin epidermis,dermis and epidermis+dermis in the IL-33 intradermal injection+model group,with obvious congestion and swelling,and the difference was statistically significant（P<0.05）.While no statistically significant difference was detected that there was no significant change in the thickness as well as congestion and swelling in mouse skin epidermis,dermis and epidermis+dermis between the IL-33 intraperitoneal injection+model group and the model group（P>0.05）.Furthermore,the results of immunohistochemistry and Western blotting revealed that compared with the model group,there was an increase in the expression of KRT14 in mouse skin of the IL-33 intradermal injection+model group,with a statistically significant difference（P<0.05）.However,with no evident change,there was no statistically significant difference in KRT14 expression between the IL-33intraperitoneal injection+model group and the model group（P>0.05）.As indicated by the results of Western blotting,there was no statistically significant difference in the expression of TRPV1 in mouse skin and spleen between the dd H2O intraperitoneal injection group and IL-33 intraperitoneal injection group（P>0.05）,between erlotinib-induced model group and IL-33intraperitoneal injection+erlotinib-induced model group（P>0.05）,between dd H2O intradermal injection group and IL-33 intradermal injection（P>0.05）,or between erlotinib-induced model group and IL-33 intradermal injection+erlotinib-induced model group（P>0.05）.Therefore,the local and systematic changes of TRPV1 expression were independent of the change in the expression of IL-33.4.EA pretreatment at Zusanli could alleviate erlotinib-induced early dermal inflammatory reaction by down-regulating the expression of TRPV1 to decrease the expression of IL-33 in mouse skin.In view of the results of HE staining,compared with the EA+model group,there was a reduced thickness in epidermis,dermis,epidermis+dermis of mice from the AMG-517+EA+model group,which,however,was increased in the Capsicine+EA+model group,with statistically significant differences（P<0.05）.Furthermore,the results of Western blotting revealed that compared with the EA+model group,the AMG-517+EA+model group had reduced expressions of TRPV1 in mouse spleen and skin,with statistically significant differences（P<0.01,P<0.05）,while Capsicine+EA+group had increased expressions when compared with those in EA+model group,with statistically significant difference（P<0.05）.Both immunohistochemistry and Western blotting results revealed that compared with the EA+model group,KRT14expression in mouse skin tissue was reduced in the AMG-517+EA+model group but increased in the Capsicine+EA+model group,with statistically significant differences（P<0.05）.Meanwhile,based on the results of immunohistochemistry,there were statistically significant differences that the AMG-517+EA+model group had reduced expression while the Capsicine+EA+model group had increased expression of LY6G in mouse skin when compared with those in the EA+model group（P<0.05）.In addition,the results of ELISA showed reduced IL-33 expression in mouse serum and skin homogenate of AMG-517+EA+model group,but increased expression of Capsicine+EA+model group than those in EA+model group（P<0.05）.Collectively,local and systematic expressions of IL-33 in mice tended to be consistent with those of TRPV1,showing the corresponding change with that of TRPV1.5.EA pretreatment at Zusanli produced no significant impact on the expression of EGFR in the erlotinib-induced model groupThe results of immunohistochemistry presented no statistically significant difference of EGFR expression in mouse skin between the erlotinib-induced model and EA+erlotinib-induced model before and after EA pretreatment（P>0.05）.Similarly,Western blotting revealed no statistically significant difference of EGFR expression in mouse skin and spleen between the two groups（P>0.05）.Hence,EA pretreatment at Zusanli for 7 days may have no significant impact on the expression of EGFR in mouse skin and spleen from the erlotinib-induced model group.Conclusions1.EA pretreatment at Zusanli can effectively inhibit the erlotinib-induced dermal inflammatory reaction.It can alleviate serum immunoglobulin production,suppress serum and skin inflammatory factor release,and inhibit KRT14 and LY6G expressions in the modeled mice,thereby relieving dermal inflammatory reaction.2.EA pretreatment at Zusanli can inhibit TRPV1 expression in mouse skin and spleen,and reduce IL-33 release in mouse serum and skin homogenate.3.EA pretreatment at Zusanli can relieve erlotinib-induced dermal inflammatory reaction by downregulating TRPV1 expression and decreasing IL-33 release of local skin in the modeled mice.4.There exists no statistically significant difference in EGFR expression of mouse skin and spleen in the erlotinib-induced model group before and after EA pretreatment.Collectively,EA pretreatment at Zusanli for 7 days may produce no impact on the efficacy of the lung cancer-targeted drug of erlotinib. |
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