Effect Of Orexin-A Microinjection Into AP On Cisplatin-induced Anorexia In Rats And Its Potential Mechanism

Objective: Cisplatin is a common drug for tumor chemotherapy,which plays an important role in many kinds of chemotherapy drugs.It has good therapeutic effect and is widely used,but there are often a series of side effects in its application,such as nausea and vomiting,kidney and liver damage,and ototoxicity.Orexin-A,also known as fat element,is mainly involved in the regulation of food intake,body weight and energy balance.Orexin-A neurons are mainly distributed in the lateral hypothalamic area?LHA?and perifornical area?PEF?of the hypothalamus,but these neurons can send out fibers to the hypothalamus,which is involved in the regulation of food intake,and can also be associated with the vagal complex?including the dorsal vagal nucleus,the solitary tract nucleus and the polar posterior region?which is involved in the regulation of gastrointestinal sensation and motility There is a fiber connection.The central injection of orexin-A in rodents can significantly promote gastrointestinal motility,increase food intake and body weight.The stimulation of orexin neurons in LHA can activate the AP neurons in the posterior area of the vagal complex?DVC?,and then promote gastrointestinal motility and pancreatic secretion.However,whether orexin-A is involved in cisplatin induced anorexia in AP has not been reported.Therefore,this study aims to observe the effect of orexin-A microinjection of AP on anorexia or anorexia induced by cisplatin in rats and its potential mechanism,so as to provide a credible experimental basis for the prevention and treatment of nausea,vomiting or anorexia caused by cisplatin.Methods: 1.Real-time PCR was used to observe the effect of intraperitoneal injection of cisplatin on the expression of Orexin-A m RNA in AP.30 rats were randomly divided into three groups?n=10?,Normal Saline control group?NS group?: intraperitoneal injection of 4 m L/kg of Normal Saline;24 h cisplatin injection group: intraperitoneal injection of 4 m L/kg of cisplatin;48 h cisplatin injection group: intraperitoneal injection of 4 m L/kg of cisplatin.To observe and compare the effect of cisplatin on the expression of AP Orexin-A m RNA.2.The effects of AP microinjection of Orexin-A on the discharge activity of cisplatin-induced gastric traction response?GD?neurons were observed by single-cell extracellular discharge recording.In this experiment,100 rats(40 NS_ip rats and 60 cisplatin_iprats)were used,GD neurons were collected and sprayed on neurons with multi-tube microelectrodes to record the changes of extracellular discharges.When the firing frequency of neurons increases or decreases by 20% before and after treatment,that is,the excitation/inhibition of neurons.3.To observe the effect of AP microinjection of Orexin-A on cisplatin-induced feeding activity and the regulation of Ghrelin receptor signaling pathway by feeding standard animal feed or kaolin.40 rats were randomly divided into four groups?n=10?:?1?NS_ip+NS_AP group: NS?4 m L/kg?was intraperitoneally injected,AP was injected NS 0.5?l;?2?cisplatin_ip+NS_AP group: intraperitoneal injection cisplatin?4 mg/kg?,AP was injected NS 0.5?l;?3?cisplatinip+Orexin-AAP group: intraperitoneal injection cisplatin?4 mg/kg?,AP was injected orexin-A?150 pmol?;?4?cisplatin_ip+Orexin-AAP+[D-Lys-3]-GHRP-6AP group: cisplatin?4 mg/kg?was intraperitoneally injected,and the mixture of Orexin-A?150 pmol?and [D-Lys-3]-GHRP-6?30 nmol?was 0.5?l was injected into AP.Cisplatin was injected intraperitoneally only once,and AP was injected once a day for 5 consecutive days.4.The effect of AP microinjection of Orexin-A on substance P in CSF induced by cisplatin was observed by ELISA.40 rats were randomly divided into four groups?n=10?:?1?NS_ip+NS_AP group: NS?4 m L/kg?was intraperitoneally injected,AP was injected NS 0.5?l;?2?cisplatin_ip +NS_AP group: cisplatin?4 mg/kg?and AP was injected NS 0.5 ?l;?3?cisplatin_ip+Orexin-A_AP group: intraperitoneal injection cisplatin?4 mg/kg?,AP was injected orexin-A?150 pmol?;?4?cisplatin_ip+ Orexin-A_AP+[D-Lys-3]-GHRP-6_AP cisplatin?4 mg/kg?was intraperitoneally injected,and the mixture of Orexin-A?150 pmol?and [D-Lys-3]-GHRP-6?30 nmol?was 0.5?l was injected into AP.All AP injections were 0.5?l.Cerebrospinal fluid was collected 24 hours after the end of the experiment and substance P content in cerebrospinal fluid was detected.Results: 1.Real time-PCR study showed that the expression of AP Orexin-A was significantly decreased after 24 or 48 hours of intraperitoneal injection of cisplatin compared with NS group?P < 0.05?,but there was no significant difference in the expression of AP Orexin-A between 24 hours of cisplatin group and 48 hours of cisplatin group?P > 0.05?.2.Single-cell electrophysiological studies showed that 120 spontaneous firing neurons were collected from the AP of 40 rats injected intraperitoneally with NS,of which 76 were gastric traction-responsive neurons?GD-N,76/120,63.3%?,including 36 gastric traction-excitatory neurons?GD-E?and 40 gastric traction-inhibiting neurons?GD-I?.In 60 rats injected with cisplatin for 24 hours,168 spontaneous firing neurons were collected from AP.Of the discharging neurons,96 were GD-N?96/168,57.1%?,including 52 GD-E and 44 GD-I.However,there was no significant difference in the percentage of GD-E and GD-I neurons between NS group and cisplatin group?P > 0.05?.Compared with rats in NS_ip + NS_AP group,the discharge frequency of GD-E neurons in cisplatin_ip+ NS_AP group increased significantly?P < 0.05?,while that of GD-I neurons decreased significantly?P < 0.05?;compared with rats in NS_ip + NS_AP group,intraperitoneal injection of NS and AP microinjection of Orexin-A significantly inhibited the discharge activity of GD-E neurons?P < 0.05?,but the discharge frequency of GD-I neurons increased significantly?P < 0.05?.Compared with cisplatin_ip+ NS_AP group,the discharge frequency of GD-E neurons in cisplatin_ip+ Orexin-AAP group decreased significantly?P < 0.05?,while the discharge frequency of GD-I neurons increased significantly?P < 0.05?.Moreover,the excitation/inhibition of GD neurons induced by Orexin-A was completely blocked by AP pre-injection of SB-334867,an Orexin-A receptor antagonist?P < 0.05?.These results suggest that cisplatin can alter the electrophysiological characteristics of GD-reactive neurons,and AP Orexin-A can suppress the excitatory changes of neurons induced by cisplatin.Further statistical analysis showed that the effect of Orexin-A on excitation/inhibition of GD neurons in NS_ip or cisplatin-ip rats was different.That is to say,compared with NS_ip+Orexin-AAP rats,the inhibition of AP injection of cisplatin_iprats on discharge activity of GD-E neurons was weakened?P < 0.05?,but the excitation of GD-I neurons was enhanced?P < 0.05?.3.The results of ELISA showed that the content of substance P in CSF of rats in cisplatin_ip+ NS_AP group was significantly higher than that in NS_ip + NS_AP group?P < 0.05?.Compared with cisplatin_ip+ NS_AP group,the content of substance P in CSF of rats in AP microinjection of Orexin-A(cisplatin_ip+ Orexin-AAP group)was significantly lower?P < 0.05?;compared with cisplatin_ip+ Orexin-AAP group,AP injection of Orexin-A + [D-Lys-3]-GHRP-6 mixed solution,the content of substance P in cerebrospinal fluid of rats increased significantly?P < 0.05?,that is,the effect of Orexin-A on reducing substance P was weakened.4.Compared with NS_ip + NS_AP group,24 hours after intraperitoneal injection of cisplatin,the intake of rats(cisplatin_ip+ NS_AP group)decreased significantly?P < 0.05?,and the effect of cisplatin-induced anorexia lasted for 5 days?P < 0.05?,and the cumulative intake decreased by 40% in 1-5 days?d?.Compared with cisplatin_ip+ NS_AP group,the intake of cisplatin_ip+ Orexin-AAP group increased significantly?P < 0.05?.These results suggest that Orexin-A can improve the anorexia induced by cisplatin.If the mixture of Orexin-A and ghrelin receptor antagonist [D-Lys-3]-GHRP-6 was injected into AP,the anorexia effect of Orexin-A against cisplatin could be partially reduced?P < 0.05?.These results suggest that the anti-anorexia effect of Orexin-A may be related to Ghrelin signaling pathway.5.The results of heterophilia induced by cisplatin showed that the cumulative daily intake of kaolin in cisplatin_ip+ NS_AP group was significantly higher than that in NS_ip + NS_AP group 1-5 days after intraperitoneal injection of cisplatin?P < 0.01?.It is suggested that cisplatin can induce heterophilia in rats?increased kaolin intake?.If the rats were injected with orexin-A(cisplatin_ip+ Orexin-AAP group)in advance,compared with cisplatin_ip+ NS_AP group,the daily cumulative intake of kaolin was significantly reduced?P < 0.05?.These results suggest that Orexin-A can improve cisplatin-induced heterophilia in rats.If AP was injected with Orexin-A+ [D-Lys-3]-GHRP-6 mixture,the resistance of Orexin-A to cisplatin-induced heterophilia could be partially reduced?P < 0.05?.These results suggest that Ghrelin receptor signaling pathway plays an important role in the improvement of cisplatin-induced side effects by Orexin-A.Conclusion: AP has gastric traction sensory afferent related neurons.Cisplatin can affect the excitability of these neurons.Orexin-A also participates in the regulation of cisplatin-induced firing activities of these neurons.AP Orexin-A can attenuate the anorexia or heterophilia induced by cisplatin.Substance P and Ghrelin receptor signaling system may also participate in the regulation of this process.