Rescue of auditory hair cells from ototoxicity by CEP-11 004, an inhibitor of the JNK signaling pathway

The hair cells (HCs) are the most vulnerable elements in the cochlea and damage to them is the most common cause of sensorineural hearing loss (SNHL). Understanding the intracellular events that lead to the death of HCs is a key to developing protective strategies. Recently, it has been shown that the c-Jun-N-terminal kinase (JNK) pathway is activated in HCs in response to aminoglycosides and CEP-1347, an inhibitor of the JNK signaling pathway protected HCs from ototoxicity. We have studied another inhibitor (CEP-11 004) of this signaling pathway in its ability to protect HCs from aminoglycoside ototoxicity in vitro. Organ of Corti explants from p5 rat basal turns were maintained in tissue culture and treated with CEP-11 004 for 12 hours. They were then treated with CEP-11 004 plus gentamicin for 72 hours. Significantly less HC death was observed compared to gentamicin alone. CEP-11 004 alone had no effect on HCs. We conclude that the JNK signaling pathway plays a role in aminoglycoside ototoxicity signaling.     

Rescue of auditory hair cells from aminoglycoside toxicity by Clostridium difficile toxin B,an inhibitor of the small GTPases Rho/Rac/Cdc42

The hair cells (HCs) are the most vulnerable elements in the cochlea and damage to them is the most common cause of sensorineural hearing loss. Understanding the intracellular events that lead to the death of HCs is a key to developing protective strategies. Recently, it has been shown that the c-Jun-N-terminal kinase (JNK) pathway is activated in HCs in response to aminoglycosides (J. Neurosci. 20 (2000) 43). We have studied the upstream events leading to JNK activation in aminoglycoside toxicity in vitro. The small GTPases Rac and Cdc42 are well known upstream activators of JNK in other cell types. Clostridium difficile toxin B monoglucosylates all members of the Rho GTPase subfamily (Rho, Rac and Cdc42 isoforms) and inhibits GTP binding by steric interference (Nature 341 (1989) 209). Organ of Corti explants from p5 rat basal turns were maintained in tissue culture and treated with C. difficile toxin B for 12 h. They were then treated with toxin B plus gentamicin for 72 h. Significantly less HC death was observed compared to with gentamicin alone. Toxin B alone had no effect on HCs at the highest concentration used. Using antibodies against phospho-c-Jun, we observed background immunoreactivity in control explants, strong staining of outer hair cell nuclei in gentamicin treated explants, and weaker immunostaining in explants treated with gentamicin and C. difficile toxin B. We conclude that Rho family small GTPases play a role in aminoglycoside toxicity signaling as upstream activators of the JNK signaling pathway.     

依达拉奉对豚鼠耳蜗急性声损伤的保护作用

目的 探讨自由基清除剂依达拉奉对豚鼠耳蜗急性声损伤的保护作用.方法 48只白色红目雌性豚鼠随机分为6组.A组(空白对照组):不做任何处置,单纯检测听功能和耳蜗自由基含量;B组:鼓室注射生理盐水;C组:鼓室注射依达拉奉;D组:单纯噪声暴露;E组:噪声暴露+静脉注射依达拉奉;F:噪声暴露+鼓室注射依达拉奉.D、E、F组在声压级125 dB的稳态噪声暴露前2 d及暴露2 h后即刻、2、6、12、24、48和72 h检测听功能和耳蜗自由基含量.听功能检测为听性脑干反应(ABR),自由基检测采用电子顺磁共振(electron spin resonance,ESR)技术.比较各组动物在不同时间点ABR阈移及自由基含量的变化.结果 急性声损伤后豚鼠ABR阈值明显升高,与空白对照组比较,差异具有统计学意义(P<0.05),暴露后72 h仍未恢复正常.鼓室注射依达拉奉可使阈值下降约10 dB,而静脉注射则无此作用.空白对照组豚鼠耳蜗自由基值为21.68(cm/g),急性声损伤后耳蜗自由基含量明显增加,在噪声暴露后2 h达峰值,至观察结束时仍未恢复正常.静脉注射依达拉奉组对噪声损伤后耳蜗自由基生成未见明显抑制作用,而鼓室注射组则可明显抑制自由基产生.结论 经鼓室局部应用依达拉奉,对豚鼠急性声损伤后的耳蜗听觉功能具有保护作用,其机制可能与有效清除局部自由基有关.     

Distribution of gentamicin by immunofluorescence in the guinea pig inner ear

We studied the distribution of gentamicin in the inner ear, brain and kidney of the guinea pig following intraperitoneal administration or perfusion of gentamicin through the perilymphatic space. The resulting histopathological changes were examined by immunofluorescence using antigentamicin antiserum. After perfusion of gentamicin through the perilymphatic space, specific fluorescence was found in the cochlea, and was especially prominent in the outer hair cells, basilar membrane and basilar crest. Although no fluorescence was observed in the cochlea following intraperitoneal administration of high doses of gentamicin, type I hair cells in the vestibule were seen to be selectively stained with the antibody. Furthermore, some of the vestibular ganglion cells, Purkinje cells and unidentified nuclei in the brain stem were also stained. In particular, fine granules showing relatively intense fluorescence were recognized in the cytoplasm of the stained cells. In the cortex of kidney, only proximal tubular cells were stained with intense fluorescence. Our results suggest that the aminoglycoside antibiotics have two sites of action: one is the cell membrane of the sensory hair cells and the other is the cytoplasm.     

庆大霉素慢性耳中毒对听觉和传出神经功能的影响

目的：探讨庆大霉素慢性耳中毒对听觉和传出神经功能的影响。方法：在庆大霉素应用前后通过观察对侧噪声（ＣＬＮ）对听神经复合动作电位（ＣＡＰ）的影响确定内侧橄榄耳蜗（ＭＯＣ）系统功能,通过测试在４,６,８,１０和１２ｋＨｚ的ＣＡＰ反应阈确定听功能。结果：注射庆大霉素后３周和１１周。ＣＬＮ对ＣＡＰ的抑制效应呈进行性、不可逆性消除,且以１１周最明显,ＣＡＰ反应阈分别上升１０和２５ｄＢ,与耳蜗传出神经和毛细胞     

Anatomical correlates of functional recovery in the avian inner ear following aminoglycoside ototoxicity

Tucci and Rubel have demonstrated functional recovery of the chick cochlea following aminoglycoside ototoxicity. The cochleae of these same animals were examined by scanning electron microscopy (SEM) in order to further understand this recovery process. Hatchling chicks were given daily doses of gentamicin for 10 days. Auditory-evoked potential measurements and examination of the cochlea by scanning electron microscopy were performed after survival periods of 5 days to 20 weeks. After 5 days of gentamicin exposure, there was near complete basal hair cell loss associated with a high-frequency hearing loss. Apical progression of damage with a broad-band hearing loss occurred over 4 weeks. At 20-weeks, hair cell counts were normal with a small high-frequency hearing loss. Hair cell regeneration played a major role in the functional recovery of the cochlea.     

庆大霉素鼓室内注射后在内耳细胞中的分布

目的 观察鼓室内注射庆大霉素后，不同时间庆大霉素在前庭和耳蜗中的分布。方法 将庆大霉素同德州红连接形成庆大霉素-德州红耦联物后，行豚鼠鼓室内注射，注射后12h，1、2、3、4、7、14、28d处死动物，Phalloidin染色后运用激光共聚焦扫描显微镜观察基底膜、椭圆囊、球囊、外半规管壶腹嵴庆大霉素分布情况，并进行荧光分布半定量分析。结果 庆大霉素自注射后12h起在内耳所有细胞均见分布，在基底膜的外毛细胞、椭圆囊、球囊、外半规管壶腹嵴的感觉细胞聚集明显，主要聚集在毛细胞顶端纤毛下方的细胞质中，支持细胞分布较少。注射后第3天庆大霉素在内耳聚集达到最高峰，并在毛细胞内聚集较长时间。结论 庆大霉素-德州红耦联物是一个研究庆大霉素在内耳分布的良好的荧光探针，可用来检测庆大霉素的药代动力学和聚集机制．     

Dependence of hearing changes on the dose of intratympanically applied gentamicin: a meta-analysis using mathematical simulations of clinical drug delivery protocols

Objectives/Hypothesis: To establish safe dosing protocols for the treatment of patients with Meniere's disease with intratympanic gentamicin. Study Design: A validated computer model of gentamicin dispersion in the inner ear fluids was used to calculate cochlear drug levels resulting from specific clinical delivery protocols. Dosing in the cochlea was compared with changes of hearing sensitivity for 568 patients reported in 19 publications. Methods: Cochlear drug levels were calculated based on the concentration and volume of gentamicin applied, the time the drug remained in the middle ear, and on the specific timing of injections. Time courses were quantified in terms of the maximum concentration (C _max) and the area under the curve of the drug at specific cochlear locations. Results: Drug levels resulting from single, “one‐shot” injections were typically lower than those from repeated or continuous application protocols. Comparison of hearing sensitivity changes with gentamicin dosing revealed a flat curve with a near‐zero mean for lower doses, suggesting that hearing changes with doses over this range were probably unrelated to the applied drug. Higher intracochlear doses were generated with repeated or continuous delivery protocols, which in some cases caused substantial hearing losses and an increased incidence of deafened ears. Conclusions: One‐shot application protocols produce gentamicin doses in the cochlea that have minimal risk to hearing at the frequencies tested. Repeated or continuous application protocols result in higher doses that in some cases damage hearing. The high variability of hearing changes, even with low gentamicin doses, calls into question the rationale for using individual hearing changes to titrate the applied dose.     

Intratympanic ototoxicity: influence of post-injection survival period

Many studies have been reported on the intratympanic ototoxicity of different drugs in animal models. The recovery periods of the animals following intratympanic drug applications varied among these studies. The present study compares the cochlear damage caused by intratympanic kanamycin following short (4 days) and long (30 days) post-injection survival periods, using the guinea pig as the animal model. The degree of cochlear damage 4 days after kanamycin injection was consistent among the tested animals. The degeneration was mainly confined to the outer hair cells and almost all inner hair were spared. The change 30 days after kanamycin injection was more variable among the animals and both inner and outer hair cells were damaged. This shows that, although the damage to the cochlea after intratympanic aminoglycoside injection is progressive, a short post-injection recovery period is suitable for comparative intratympanic ototoxicity studies.     

Effect of intratympanic dexamethasone administration on cisplatin-induced ototoxicity in adult guinea pigs

Objective

To evaluate the effect and safety of intratympanic dexamethasone administration on cisplatin-induced ototoxicity in adult male guinea pigs and to assess the differences between early and late protection from this ototoxicity.

Methods

Forty eight adult male guinea pigs were divided as follows: group I served as control group. Group II was subjected to intratympanic saline (subgroup IIa) or dexamethasone (subgroup IIb) injection. Group III was intraperitoneally injected with cisplatin. Groups IV and V were subjected first to intratympanic dexamethasone administration in both ears for 5 days starting 1 day and 1 h – respectively – before cisplatin intraperitoneal injection.

Results

Dexamethasone intratympanic injection revealed similar functional and structural results compared with control. Cisplatin intraperitoneal injection resulted in a profound cochlear functional and structural damage in group III. Non-significant otoprotection resulted from intratympanic dexamethasone administration one day before cisplatin. Intratympanic dexamethasone injection 1 h before cisplatin treatment resulted in a significant preservation of the functional and structural properties of the cochlea.

Conclusion

Intratympanic dexamethasone administration is a safe, easy and efficient way to protect from cisplatin ototoxicity especially when administered 1 h before cisplatin treatment.     

庆大霉素对内侧橄榄耳蜗传出神经毒性作用的形态学观察

目的 观察庆大霉素慢性耳中毒前后蒙古沙鼠人侧橄榄耳蜗传出神经的形态改变及其与细胞损害的关系,以探讨ＭＯＣ传出神经在氨基糖甙类抗生素慢性耳中毒中的重要性。方法 采用改良的乙酰胆碱酯酶组化染色和甲苯胺蓝＝苏木素染色法,全耳蜗铺片观察健康对照组和庆大霉素组耳蜗ＭＯＣ传出神经和外毛细胞的分布特征,并测量耳蜗ＭＯＣ传了神经纤维、末梢及ＯＨＣ的数量。结果 停经后３周出现耳蜗ＭＯＣ纤维和末梢损害,损害程度随时间     

Comparison of noninvasive evaluation of endolymphatic space in healthy volunteers in different age groups using magnetic resonance imaging

Conclusions: Noninvasive standard evaluation of normal endolymphatic space and endolymphatic hydrops using magnetic resonance imaging (MRI) in various age groups is reported for the first time. Objective: To compare the standard evaluation of endolymphatic space in healthy volunteers in the cochlea and the vestibule among different age groups by applying noninvasive intratympanic gadolinium (Gd) perfusion through the eustachian tube and three-dimensional fluid-attenuated inversion recovery MRI (3D-FLAIR MRI). Methods: This was a prospective study. 3D-FLAIR MRI was performed with a 3 T unit 24 h after intratympanic administration of Gd through the eustachian tube in 60 healthy volunteers aged 20–55 years. Pure-tone test and tympanometry were performed 24 h before and 1 week after Gd administration. Results: There was no significant difference in the ratios of the area of the endolymphatic space to that of the fluid space in the cochlea and the vestibule between males and females, or among 20–30-, 31–44-, and 45–55-year-old healthy volunteers. In 20–55-year-old healthy volunteers, the normal value of the endolymphatic space in the cochlea ranged between 7% and 27%, and that in the vestibule was between 17% and 39%. No significant changes in pure-tone test or tympanometry were noted.     

Comparisons of cochleotoxicity among three gentamicin compounds following intratympanic application

CONCLUSION: Among the three main gentamicin (GM) compounds following intratympanic application, the cochleotoxicity of C2 was the most severe, whereas that of C1a was the weakest. Understanding of the different cochleotoxicity characteristics of each compound may be of use in future custom-made intratympanic therapy for Ménière's disease. OBJECTIVE: To investigate differences in cochleotoxicity among three major GM compounds following intratympanic application. MATERIALS AND METHODS: Three GM compounds (C1, C2, and C1a) were isolated. Sprague-Dawley rats were treated every 2 days for 2 weeks with intratympanic application of saline, GM complex, C1, C2, and C1a. The cochleotoxicity of each compound was assessed by measuring auditory brainstem response (ABR) and through morphological analyses using scanning electron microscopy. RESULTS: The ABR threshold of the C2 group was found to be more impaired than those of the other groups. The C1a group showed the mildest elevation of the ABR thresholds. Morphological analyses revealed that the proportion of remaining outer hair cells (OHCs) was the lowest in animals treated with C2. Morphologically, the C1 and C1a groups showed the least damage to OHCs.     

Treatment of Ménière's disease by low-dosage intratympanic gentamicin application: effect on otolith function

OBJECTIVES: The intratympanic application of a low dosage of gentamicin is increasingly favored as treatment for Ménière's disease. While posttreatment observations have confirmed a long-term success of the therapy of vertigo attacks, clear differences in the posttreatment recovery interval can be observed. In addition to differences in central-vestibular compensation, the degree of peripheral vestibular damage, i.e., to the saccule, utricle, and semicircular canal ampullae, varies among patients. This study provides comprehensive pre- and posttreatment results from unilateral functional tests of the individual vestibular receptors and of the cochlea in patients with Ménière's disease. STUDY DESIGN: Prospective clinical study. METHODS: Nineteen patients with unilateral Ménière's disease were treated by intratympanic application of gentamicin by injection of 0.3 mL (12 mg) through the tympanic membrane under local anesthesia. Tests were performed immediately previous to treatment and subsequently in the periods 4 to 8 weeks and 12 to 16 weeks after treatment. Unilateral saccular function was tested by means of acoustic-click, vestibular-evoked myogenic potentials (VEMP), and unilateral utricular function by subjective visual vertical (SVV) during unilateral centrifugation. Bithermal caloric testing was performed to assess unilateral semicircular canal function. RESULTS: Prior to gentamicin treatment, the caloric response from the diseased ear was normal in 3 patients, below normal in 14 patients, and in 2 cases almost completely absent. VEMP responses could be recorded bilaterally in 13 patients; while in 6, no VEMPs could be measured from the diseased ear. Utricular function measured by SVV estimation was found to be normal in 11 patients and marginally abnormal in 2 patients. In six cases, the SVV was clearly underestimated during centrifugation of the diseased side. The posttreatment findings demonstrate that VEMPs were absent in all treated patients, and the caloric response was abnormally low in all but one case. In contrast, only 12 of 19 patients produced abnormal SVV responses. CONCLUSION: The results demonstrate that incremental, intratympanic application of gentamicin effectively eliminates semicircular canal and saccular function. In contrast, utricular function appears to be maintained in 30 to 40% of cases.     

Distribution of gentamicin by immunofluorescence in the guinea pig inner ear

Summary We studied the distribution of gentamicin in the inner ear, brain and kidney of the guinea pig following intraperitoneal administration or perfusion of gentamicin through the perilymphatic space. The resulting histopathologcial changes were examined by immunofluorescence using antigentamicin antiserum. After perfusion of gentamicin through the perilymphatic space, specific fluorescence was found in the cochlea, and was especially prominent in the outer hair cells, basilar membrane and basilar crest. Although no fluorescence was observed in the cochlea following intraperitoneal administration of high doses of gentamicin, type I hair cells in the vestibule were seen to be selectively stained with the antibody. Furthermore, some of the vestibular ganglion cells, Purkinje cells and unidentified nuclei in the brain stem were also stained. In particular, fine granules showing relatively intense fluorescence were recognized in the cytoplasm of the stained cells. In the cortex of kidney, only proximal tubular cells were stained with intense fluorescence. Our results suggest that the aminoglycoside antibiotics have two sites of action: one is the cell membrane of the sensory hair cells and the other is the cytoplasm.This study was supported in part by a grant from the Ministry of Education, Science and Culture of Japan, and by a Research Grant for Specific Diseases from the Ministry of Health and Welfare to the Acute Profound Deafness Research Committee of Japan     

Changes in the avian cochlea after single high-dose gentamicin

PURPOSE: Define the time course of functional and anatomical damage and subsequent recovery (by regeneration) of hair cells in the chicken inner ear after a single high-dose of gentamicin. MATERIALS AND METHODS: Broiler chicks were given a single intraperitoneal dose (200 mg/kg) of gentamicin (n = 39) or saline (n = 39). Functional status was evaluated with auditory brainstem response (ABR) thresholds before injection and before sacrifice at 2, 5, 9, 16, 21, 28, and 70 days postinjection. The cochleae were then examined with scanning electron microscopy (SEM) to assess the extent of damage along the cochlea and absolute hair cell numbers in the basal 15% of the cochlea (high-frequency region). RESULTS: Considerable variability between animals was seen for both ABR and SEM changes. Damage was maximal at 5 days postinjection with an average ABR threshold shift of 12 dB (range -10 to 50 dB) and basal cochlear damage of 28% (range 12%-57%). Hair cell counts were significantly decreased in the basal 15% of the cochlea at 5 days. Hair cell regeneration resulted in rapid anatomical and functional recovery, but evidence of hair cell disorganization persisted at 70 days despite improved thresholds. CONCLUSION: A single high dose of gentamicin produces a significant but variable anatomical and functional insult in the chick cochlea. Hair cell regeneration results in rapid but incomplete recovery.     

Intratympanic Gentamicin for Treatment of Intractable Meniere's Disease: A Preliminary Report

Topical administration of aminoglycoside antibiotics in the middle ear can achieve “chemical labyrinthectomy” in patients with intractable Meniere's disease. Herein we report our results of intratympanic gentamicin therapy in 21 patients using two different dosing protocols, twice weekly and twice daily(b.i.d.). Both hearing and vertigo outcome were evaluated. Complete control of episodic vertigo was achieved initially in 20 of 21 patients (95.2%). However, 6 of 20 responders (30%) developed relapsing symptoms within 12 months. Retreatment was successful in 75% of these patients. Overall, hearing was preserved or improved in 62% of cases, worse in 24%, and not yet tested in 14%. When the cumulative dose of gentamicin was ≤4 injections in the first week, only 1 of 14 (7.1%) lost hearing. Intratympanic gentamicin offers better risk/benefit outcome than other invasive therapies for intractable Meniere's disease.     

Hair cell regeneration after local application of gentamicin at the round window of the cochlea in the pigeon

Hair cells in the basilar papilla of birds have the capacity to regenerate after injury. Methods commonly used to induce cochlear damage are systemic application of ototoxic substances such as aminoglycoside antibiotics or loud sound. Both methods have disadvantages. The systemic application of antibiotics results in damage restricted to the basal 50% of the papilla and has severe side effects on the kidneys. Loud sound damages only small parts of the papilla and is restricted to the short hair cells. The present study was undertaken to determine the effect of local aminoglycoside application on the physiology and morphology of the avian basilar papilla. Collagen sponges loaded with gentamicin were placed at the round window of the cochlea in adult pigeons. The time course of hearing thresholds was determined from auditory brain stem responses elicited with pure tone bursts within a frequency range of 0.35–5.565 kHz. The condition of the basilar papilla was determined from scanning electron micrographs. Five days after application of the collagen sponges loaded with gentamicin severe hearing loss, except for the lowest frequency tested, was observed. Only at the apical 20% of the basilar papilla hair cells were left intact, all other hair cells were missing or damaged. At all frequencies there was little functional recovery until day 13 after implantation. At frequencies above 1 kHz functional recovery occurred at a rate of up to 4 dB/day until day 21, beyond that day recovery continued at a rate below 1 dB/day until day 48 at the 5.6 kHz. Below 1 kHz recovery occurred up to day 22, the recovery rate was below 2 dB/day. A residual hearing loss of about 15–25 dB remained at all frequencies, except for the lowest frequency tested. At day 20 new hair cells were seen on the basilar papilla. At day 48 the hair cells appeared to have recovered fully, except for the orientation of the hair cell bundles. The advantage of the local application of the aminoglycoside drug over systemic application is that it damages almost all hair cells in the basilar papilla and it has no toxic side effects. The damage is more extensive than with systemic application.     

Doxycycline reduces nitric oxide production in guinea pig inner ears

Objective

Gentamicin application is an important therapeutic option to control vertigo spells in Ménière's disease. However, even in the case of low-dose intratympanic application, gentamicin might contribute to a pathological NO-increase leading to cochlear damage and hearing impairment. The study was performed to evaluate the nitric oxide (NO) reducing capacity of doxycycline in the inner ear after NO-induction by gentamicin.

Methods

In a prospective animal study, a single dose of gentamicin (10 mg/kg body weight) was injected intratympanically into male guinea pigs (n = 48). The auditory brainstem responses (ABRs) were recorded prior to application and 3, 5 and 7 days afterwards. The organ of Corti and the lateral wall of 42 animals were isolated after 7 days and incubated separately for 6 h in cell culture medium. Doxycycline was adjusted to organ cultures of 5 animals. Two NOS inhibitors, N^G-Nitro-l-arginine methyl ester (l-NAME) and NG-monomethyl-l-arginine monoacetate (l-NMMA), were applied in three different concentrations to the organ cultures of 30 animals in total (5 animals per concentration). As controls, seven animals received no further substance except gentamicin. The NO-production was quantified by chemiluminescence. Additional six gentamicin-treated animals were used for immunohistochemical studies.

Results

The ABRs declined continuously from the first to the seventh day after gentamicin application. Doxycycline reduced NO-production in the lateral wall by 54% (p = .029) comparable to the effect of the applied nitric oxide inhibitors. In the organ of Corti, NO-production was reduced by about 41% showing no statistical significance in respect to great inter-animal variations.

Conclusion

The application of doxycycline might offer a new therapeutic approach to prevent NO-induced cochlea damage through ototoxic substances.     

庆大霉素对内侧橄榄耳蜗传出神经毒性作用的形态学观察

目的观察庆大霉素慢性耳中毒前后蒙古沙鼠内侧橄榄耳蜗（ｍｅｄｉａｌｏｌｉｖｏｃｏｃｈｌｅａｒ,ＭＯＣ）传出神经的形态改变及其与毛细胞损害的关系,以探讨ＭＯＣ传出神经在氨基糖甙类抗生素慢性耳中毒中的重要性。方法采用改良的乙酰胆碱酯酶组化染色和甲苯胺蓝苏木素染色法,全耳蜗铺片观察健康对照组和庆大霉素组耳蜗ＭＯＣ传出神经和外毛细胞（ｏｕｔｅｒｈａｉｒｃｅｌ,ＯＨＣ）的分布特征,并测量耳蜗ＭＯＣ传出神经纤维、末梢及ＯＨＣ的数量。结果停药后３周出现耳蜗ＭＯＣ纤维和末梢损害,损害程度随观察时间延长而加重,１１周最明显,且损害部位主要在耳蜗底回,与毛细胞损害的特征一致。结论耳蜗ＭＯＣ传出神经可能在庆大霉素慢性耳中毒时ＯＨＣ的损害中起重要作用。