Protective Effects And Mechanisms Of Ginsenoside Rd On Structural And Functional Damage Of Auditory System Caused By Military Helicopter Noise

The prevention and control of noise-induced injury has been a highly debated topic in society,which is of particular concern for pilots and those working in the aviation operating sector.At present,the most common method of prevention of noise-induced injury is by the use of hearing protection devices,which can reduce the damage to the auditory system.However,due to the poor compliance and discomfort of the protective devices,air and ground crew of aviation units are often unable or unwilling to wear these protective devices at work.As a result,their auditory systems are vulnerable to damage,and the incidence of auditory system diseases,such as noise-induced hearing loss,tinnitus,and hearing allergy,is high.Therefore,it is necessary to identify other means to prevent noise-induced auditory damage to complement to the use of hearing protection device to order to reduce the incidence of noise-induced hearing loss among aviation operators.Ginsenoside is one of the active components in Panax ginseng and Panax notoginseng.Ginsenoside Rd(GSRd)is a rare monomer of ginsenoside,but its pharmacological activity is better than that of main saponins.GSRd can scavenge free radicals and exert anti-oxidation,anti-aging,and analgesic effects.In addition,GSRd can provide protective effects on the cardiovascular,kidney,immune,and nervous systems.However,it has not been reported whether ginsenoside Rd has protective effect on noise-induced damage,especially the auditory system damage caused by high-intensity specific frequency noise in military aviation.ObjectiveWe aimed to establish an animal model of helicopter-noise-induced injury via continuous exposure to helicopter noise,and to observe the protective effect of ginsenoside Rd(GSRd)against the structural and functional damage to the auditory system.We also aimed to investigate GSRd’s mechanism of action to identify potential prevention and treatment options for noise-induced hearing loss.MethodsIn the first part,an animal model of helicopter-noise-induced injury was established.Thirty-five male guinea pigs were randomly divided into 5 groups:control(Con),95 dB(A),105 dB(A),115 dB(A),and 120 dB(A).The control group was not given noise stimulation.The noise used in the experiment was from the engine of a type of aircraft in the Chinese aviation force.The animals were stimulated 4 hours each day for 5 days.Auditory brainstem response(ABR),click short sound stimulation,and distortion product otoacoustic emission(DPOAE)were used to measure the hearing of guinea pigs before and after noise stimulation.To explore the protective effect of GSRd on noise-induced auditory cortex and inner ear injury,forty-eight guinea pigs were randomly divided into the control(Con)group,noise(NE)group,experimental(Rd)group,which received GSRd dissolved in glycerin through an intraperitoneal injection at a dose of 30 mg/kg body weight per day from five days before noise exposure until the end of the noise exposure period,and vehicle group(Vehl)which received glycerin through an intraperitoneal injection at a dose of 30 mg/kg body weight for the same time as the Rd group.With the exception of the control group,the guinea pigs were stimulated with recorded sound at 115 dB(A)for 4 hours a day for 5 days.The hearing levels were examined by the ABR and DPOAE tests.Hematoxylin-eosin(HE)and Nissl staining were used to examine the neuron morphology.Inner ear basilar membrane preparation was used to evaluate the degree of hair cell damage and loss.The damage to the cilia of cochlear hair cells was examined using scanning electron microscopy.The TUNEL assay and the expression of Bax and Bcl-2 were used to investigate the degree of apoptosis in the auditory cortices and inner ear cells.The expression levels of genes and proteins related to SIRT1/PGC-1α signaling pathway and of free radical 4-HNE and 3-NT in cochlea were observed.Superoxide dismutase(SOD),malondialdehyde(MDA),and glutathione peroxidase(GSH-Px)levels were determined using a commercial testing kit.ResultsIn the first part,the results of ABR test showed that the hearing threshold of animals in the 95 dB(A)and 105 dB(A)groups gradually recovered at 3 to 5 days after noise stimulation,while that in the 115dB(A)group gradually recovered at 5 to 7 days.The hearing threshold of the 120 dB(A)group did not decrease to the basic level 7 days after stimulation.The DPOAE amplitude in animals stimulated with 95 dB(A)returned to normal 5 days after the stimulation,while the amplitude in those stimulated with 105 and 115 dB(A)returned to normal 7 days after the stimulation.In the 120 dB(A)group,the amplitude of DPOAE in the low frequency band recovered 7 days after stimulation,but the amplitude in the high frequency band remained lower than the starting value.In the second and third parts,the results of ABR test showed that after noise stimulation,the hearing thresholds of the NE,Rd,and Vehl groups were significantly higher than that of the Con group(P<0.01),and the hearing threshold of the Rd group was lower than that of the NE and Vehl groups(P<0.01).The results of the DPOAE test showed that the signal amplitude of the Rd group was significantly lower than that of the NE and Vehl groups at each test frequency(P<0.01).The results of HE,Nissl and immunohistochemical staining showed that the proportion of neuronal apoptosis was lower in the Rd group compared with the NE and Vehl groups(P<0.01).There were obvious losses of hair cells and disordered arrangement of the residual cilia in the NE and Vehl groups,which were partially prevented by Rd treatment.The TUNEL positive cells were mainly distributed in the Corti’s organ,stria vascular,and spiral ganglion.The hair cell loss and cilia injury of cochlear inner hair cells in the Rd group were significantly less than those in the NE and Vehl groups,and the expression of TUNEL positive cells decreased.Rd treatment decreased the immunofluorescence expression of free radical markers 4-HNE and 3-NT induced by noise exposure in the inner ear(P<0.01).Compared with the control group,the expressions of SIRT1,PGC-1α and Bcl-2 mRNA and protein in the auditory cortex and inner ear of the NE,Rd and Vehl groups were significantly decreased(P<0.05).The expression of Bcl-2 mRNA and protein in the Rd group was significantly higher than that in the NE and Vehl groups(P<0.05).The expressions of SIRT1,PGC-1α and Bcl-2 mRNA and protein in the Rd group were significantly higher than those in the NE and Vehl groups(P<0.05),while the expression of Bax was significantly decreased(P<0.05).The contents of SOD and GSH-Px in auditory cortex and inner ear in the NE,Rd and Vehl groups were significantly lower than those in the control group(P<0.01),while the content of MDA in the NE and Vehl groups was significantly lower than that in the NE and Vehl groups(P<0.01).The contents of SOD and GSH-Px were significantly higher(P<0.05)and the content of MDA in NE group was significantly lower(P<0.05)in the Rd group than that in the NE and Vehl groups.ConclusionA 115 dB(A)military helicopter noise stimulation produces a stable sound injury model.Noise exposure can damage the structure and function of the auditory system.GSRd has a protective effect against noise-induced structural and functional damage to the guinea pig auditory system.The possible mechanism is to activate SIRT1/PGC-1α pathway,upregulate the expression of SIRT1/PGC-1α,increase the activities of SOD and GSH-Px,decrease the level of MDA,and enhance the antioxidant capacity of the auditory system,so as to reduce the structural and functional damage of auditory system caused by military helicopter noise.