Background and aims:Kehuang(KH)capsule is an herbal medical product approved for the treatment of liver diseases,including liver injury,in China.However,the mechanism is still unclear.This study aimed to elucidate the protective effects of KH capsule against carbon tetrachloride(CCl4)-induced acute liver injury(ALI)in a murine model.Methods:Mice were randomly divided into control,model(CCl4),CCl4+KH_Low and CCl4+KH_High group.Liver enzyme levels and histological changes were assessed to evaluate liver injury.Oxidative stress markers and inflammatory cell infiltration in liver tissues were measured.Additionally,network pharmacology was employed to explore the potential mechanisms of KH capsule.Results:KH capsule significantly reduced serum alanine aminotransferase(ALT)and aspartate amino-transferase(AST)levels,as well as the necrotic area in liver tissue.KH capsule also decreased the infil-tration of macrophages and neutrophils,thereby inhibiting the expression of interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α),and interleukin-1 beta(IL-1β).Furthermore,KH capsule decreased liver malondialdehyde(MDA)levels and increased superoxide dismutase(SOD)activity.The number of ter-minal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL)-positive cells in liver tissue was also reduced.The expression of nuclear factor erythroid 2 related factor 2(Nrf2)and heme oxygenase-1(HO-1)proteins was significantly elevated,while the protein expression of cyto-chrome P450 2E1(CYP2E1)was significantly reduced.Mass spectrometry identified genistein,galangin,wogonin,skullcapflavone Ⅱ,and hispidulin as potential active ingredients of KH capsule.Network pharmacology analysis revealed enrichment in the mitogen-activated protein kinase(MAPK)and phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT)signaling pathways.Western blot analysis confirmed that KH capsule suppressed AKT,extracellular signal-regulated kinase(ERK),and p38 signaling.Conclusions:These findings suggest that KH capsule could exert protective effects against CCl4-induced ALI,with the inhibition of MAPK and PI3K-AKT signaling pathways playing a crucial role in its mecha-nism of action.

Objective:To investigate the effects of Ligustrazine (TMP) on the expression of caspase-3 and ABR threshold in guinea pig cochlea after gentamicin (GM) ototoxicity. Methods:A total of 80 guinea pigs were randomly divided into saline group, GM group, GM+TMP group and TMP group. The duration of treatment was lasted 10 d continuously in all groups. The dose was decided by their weight. The ABR threshold were measured before and after administration of 10 d. SABC immunohistochemical method was used to detect the expression of caspase-3 in the spiral ganglion,cochlea hair cells,and stria vascularis of guinea pigs. Results:(1) The average gray value in the spiral ganglion cells, stria vascularis and hair cells in GM group had significant difference with those in the same position of control group (P<0.05) .The average gray value in the spiral ganglion cells and hair cells in GM+TMP group had no difference with those in the same position of control group (P>0.05), but in stria vascularis (P<0.05) . Meanwhile the average gray value in spiral ganglion, hair cells, stria vascularis in TMP+GM group had significant difference with those in GM group (P<0.05) . (2) The ABR threshold had no significant differences before treatment (P>0.05) . After administration of 10 d, the ABR threshold of GM group was significantly changed (P<0.05) . The ABR thresholds in GM group was significantly higher than that in control group (P<0.05) . The ABR threshold in TMP+GM group was lower than that in GM group (P<0.05) . Conclusion:TMP participates in preventing the process of cochlea injury caused by GM,through blocking the expression of caspase-3 in cochlea tissue of GM ototoxicosis probably,which achieve the protection of GM deafness eventually.

BACKGROUND: Formerly, it was thought that the damaged hair cells could not have the repair ability. Recent studies demonstrate that mammal vestibule hair cells also possess certain repair ability after being destroyed.Then, whether mammalia animal cochlea hair cells possess regenerative ability after being destroyed is disputed.OBJECTIVE: To observe cochlear hair cells condition and threshold value change of auditory brainstem response (ABR) at different time following gentamicin ototoxicity by using scanning electron microscope (SEM) technique combined with ABR test, so as to investigate whether cochlear hair cells of mammals can be regenerated after being injured.DESIGN: A randomized and controlled animal experiment.SETTING: Department of Physiology, Shenyang Medical College.MATERIALS: This experiment was carried out at the Hearing Research Room of China Medical University from November 2001 to May 2002. Totally 60 healthy adult white Guinea pigs, with red eyes and sensitive auricle reflex, of clean degree, were used and randomly divided into gentamicin group and normal control group with 30 guinea pigs in each one.METHODS: 100 mg/kg gentamicin was intraperitoneally daily injected into the guinea pigs, serving as gentamicin group. Same volume of normal saline (2.5 mL/kg) was intraperitoneally daily injected into the guinea pigs,serving as normal control group. All the guinea pigs were given medication for 10 successive days. Threshold value of ABR was detected respectively pre-operatively and at the 1st, 3rd, 30th days postoperatively; after withdrawal and execution, scanning electron microscope was used to observe cochlear hair cells of guinea pigs in each group.MAIN OUTCOME MEASURES: ① Threshold value of ABR. ②Cochlear hair cell change of guinea pigs at different time following gentamicin ototoxicity.RESULTS: All the 60 experimental animals entered the stage of result analysis with no loss in the midway. ①At 1,3 and 30 days after withdrawal of gentamicin, threshold value of ABR was significantly higher as compared with normal control group, with significant difference [(38.00±3.75), (2.22 ±3.63) dB nHL,t=30.651, P ＜ 0.001];[(39.09±4.22), (2.50±3.54) dB nHL, t=29.708, P ＜ 0.001];[(14.50±3.69), (1.50±2.42) dB nHL,t =13.175, P ＜ 0.001]. Threshold value of ABR recovered obviously on day 30, but did not reach the normal level. ②On the first day after withdrawal of gentamicin , stereocilium of hair cells in second turn of cochlea of guinea pigs presented fusion, distortion, lodging, loss or incompetence and other pathological changes , especially severe in the third turn , also cystic form protrutions appeared outside the stereocilium of inner hair cell; On day 3 after withdrawal of gentamicin, stereocilium of outer hair cells in the second turn of cochlea of guinea pigs still presented fusion, loss, lodging and other pathological changes. Stereocilium of inner hair cell still showed lodging,but the outside cystic form protrutions decreased; On day 30 after withdrawal of gentamicin, stereocilium of outer hair cells in the second turn of cochlea presented fusion, loss , lodging and other pathological change ,which were obviously weaker than those on the 1st day and 3rd day after withdrawal of gentamicin , and at the same time , new born stereocilium appeared in the third turn of cochlea.CONCLUSION: Cochlear hair cell morphology recovery appears in those which survive for 30 days after cochlear hair cells of guinea pigs are damaged following gentamicin ototoxicity, and threshold value of ABR also recovers to some extent, suggesting that cochlear hair cells possess regenerative and repair ability following gentamicin ototoxicity. Hair cells after gentamicin-induced cochlear damage possess regenerative ability.

BACKGROUND: Ligustrazine possesses the effect to reduce ototoxicity of gentamicin, whether does it antagonize the ototoxicity of gentamicin through influencing the expression of heat shock protein (HSP) 70 in cochlea of guinea pigs with gentamicin induced ototoxicity? BJECTIVE: To investigate the effect of ligustrazine on the expression of HSP70 in cochlea of guinea pigs with gentamicin induced ototoxicity through immunohistochemistry and image analysis technique in combination with the measurement of auditory brainstem response (ABR).DESIGN: A randomized controlled study, and linear correlation analysis. ETTING: Physiological Department of Shenyang Medical College and Audiological Laboratory of China Medical University.MATERIALS: Totally 40 white and red-eye healthy guinea pigs of lean grade and with keen auricle reflect, weighing 200 - 250 g, of either gender, were at random divided as gentamicin group, ligustrazine + gentamicin group, ligustrazine group, and normal control group, with 10 in each group.METHODS: Ligustrazine injection 140 mg/kg was intraperitoneally at the left side given for animals in ligustrazine + gentamicin group, and at the same time gentamicin sulfate injection 100 mg/kg was given intraperitoneally at the right side. The same dosage of gentamicin sulfate injection was intraperitoneally given for animals in gentamicin group. The same dosage of ligustrazine was intraperitoneally give for animals in ligustrazine group. The normal saline 2.5 mL/kg was intraperitoneally given for animals in normal control group. Administration was consecutively given for 10 days for each group, once a day. The body mass was measured every day for regulating the dosage. Before starting and after finishing the administration, the thresholds of ABR of all animals in each group were measured respectively. After finishing administration, all animals in each group were put to death, and the conditions of expressions of HSP70 in cochlea of guinea pigs were investigated by SABC immunohistochemistry and image analysis technology.MAIN OUTCOME MEASURES: ① the thresholds of ABR of all animals in each group; ② expressions of HSP70 in cochlea of guinea pigs in each group.RESULTS: ① The thresholds of ABR of guinea pigs: The thresholds in ligustrazine + gentamicin group and gentamicin group were obviously higher than that in normal control group [(21.09±4.50) dBnHL, (36.55±6.13)dBnHL, (2.50±2.75) dBnHL, t=15.764-22.665, P ＜ 0.001]. The threshold in ligustrazine + gentamicin group was obviously lower than that in gentamicin group (t=9.092, P ＜ 0.001). ② The expressions of HSP70 in each part of cochlea of guinea pigs: The mean gray values of cellular HSP70 positive reaction products in Corti's organ, vascular stria and spiral ligament, spiral limbus and spiral ganglion in gentamicin group were lower than those in normal control group (88.24±4.34, 96.85±1.05; 121.24±0.92,128.76±1.59; 96.15±1.10, 98.78±0.54; 117.73±1.18, 120.51±0.80, t=6.097-18.307, P ＜ 0.001). The mean gray values of cellular HSP70 positive reaction products in ligustrazine + gentamicin group were lower than those in gentamicin group (92.53±2.25, 88.24±4.34; 125.20±1.43, 121.24±0.92;98.71±0.91, 96.15±1.10, 118.91±0.46, 117.73±1.18, t=3.925-10.415, P＜ 0.001).③ The mean gray value of cellular HSP70 positive reaction products in each part of cochlea of guinea pigs was highly correlated with the threshold of ABR (r=-0.814 1 to -0.984 1, P ＜ 0.001).CONCLUSION: Ligustrazine could reduce the threshold of ABR and the expression of HSP70 in cochlea with gentamicin toxicosis so as to relieve gentamicin ototoxic injury, hence improving auditory function.