| Background: Parkinson's disease(PD)is the second most common neurodegenerative disease,which is mainly characterized by symptoms of tremor,rigidity and postural abnormalities.The pathological feature of the disease is the selective and progressive loss of dopaminergic neurons in the substantia nigra pars compact(SNpc)in the ventral midbrain and the presence of Lewy bodies in the degenerated dopaminergic neurons.?-synuclein is a major component of Lewy body.PD is therefore defined as a multiple system disease with Lewy neurites and Lewy bodies.Histamine is an autacoid and inflammatory mediator.Histamine plays a very important role on the regulation of allergy and inflammation in peripheral tissues and organs.Until the 1970 s,as an important neurotransmitter or neuromodulator in the central nervous system(CNS)histamine exerts its functions through its four types(H1-H4 receptor)of receptor.Previous studies have shown that histamine system in PD is in the over-activated state,and H1 R and H2 R antagonists can protect dopaminergic neurons in PD,but the role of histamine H3 R and H4 R receptors in the SNpc of PD is rarely reported.In order to clarify the effect and related mechanism of H3 R and H4 R in PD,rotenone-induced PD rats were used as a PD animal model in the present study.The effects of H3 R and H4 R antagonists on dopaminergic neurons in PD rats were investigated.After that,the protection mechanism related microglia of H4 R antagonist on dopaminergic neurons in PD was further studied.In addition,to de-termine the direct effect of H4 R antagonist on dopaminergic neurons in PD,the effect of H4 R antagonist on the damage of rotenone-induced SH-SY5 Y cell was studied as well.Methods: 1.Model preparation and drug administration: 36 adult male SD rats were injected 12 ?g rotenone in the right SNpc and an intubation was imbedded into lateral ventricular with stereotaxic apparatus.H4 R antagonist JNJ7777120(JNJ),H3 R antagonist Carcinine,and M1 subtype microglia inhibitor Donepezil(icv,5?g/day,once a day)were administration,respectively,for 3 weeks.2.Grouping,turning behavior and sample testing: Animal experiments were carried out in two big groups.In the first big group,animals were randomly divided into Rot and Rot + the 3 drug intervention groups.After three weeks of administration,the animals were decapitated and received a heart perfusion with 4% paraformaldehyde.Then,brain tissues were moved out and used for immumohistochemical staining of Tyrosine Hydroxylase(TH),ionized calcium binding adapter molecule-1(IBA-1)and ?-synuclein.In the second big group: Since the H3 R antagonist did not show any significant effect,control,Rot and Rot + H4 R antagonist groups were included for the further study.Animals were received an apomorphine-induced turning behavior per week.After three weeks of administration,the animals were decapitated and striatum and ventral midbrain were separated and preserved in-80?.The content of dopamine(DA)in the striatum of animals was detected by high performance liquid chromatography-mass spectrometry(HPLC-MS/MS).The expressions of H4 R,histamine N-methyltransferase(HMT),cluster of differentiation 86(CD86),interleukin-1?(IL-1?),cluster of differentiation 206(CD206),arginase-1(Arg1)in the ventral midbrain were detected by real-time fluorescence quantitative PCR(q PCR).3.SH-SY5 Y Cell experiment: SH-SY5 Y cells were treated with 100 nM rotenone for 72 hours.The effect of H4 R antagonist JNJ on Rot-induced decreased cell viability in SH-SY5 Y cells were detected by CCK assay.At the same time,the expression of ?-synuclein in SH-SY5 Y cells was detected by immunocytochemical staining and Western blotResults: 1.In the immunohistochemical staining: The TH positive neurons in the SNpc on the lesioned side in the Rot group were significantly reduced to 43% of unlesioned side(P<0.05).Compared with the Rot group,H4 R antagonist JNJ significantly increased the number of TH positive cells(an increase of 160%,P<0.05);while H3 R antagonist Carcinine did not show any significant effect on the number of TH positive cells.2.In the apomorphine-induced turning behavior,compared with the Rot group,H4 R antagonist JNJ significantly reduced the number of rotations(57%,41%,and 71% of Rot group at 1-3 week,respectively,P<0.05).Similar to TH staining,H4 R antagonist JNJ significantly increased the content of DA in the striatum as well(an increase of 88%,P<0.05).In addition,H4 R antagonist markedly decreased the increased expression of H4 R and HMT induced by Rot(a decrease of 4.9 and 1.9 times of Rot group)3.There were a lot of positive ?-synuclein bodies in the SNpc on the lesioned side in the Rot group,which was 210% of unlesioned side(P<0.05).H4 R antagonist JNJ significantly reduced the number of ?-synuclein body(a decreased of 34%,P<0.05).4.The number of IBA-1 positive cells,microglia marker protein,in the SN on the lesioned side in the Rot group were significantly increased,which was 222% of unlesioned side(P<0.05).H4 R antagonist JNJ significantly decreased the number of IBA-1 positive cells(41% of the Rot group,P < 0.05).The results of q PCR showed that the H4 R antagonist JNJ significantly inhibited the expression of M1 subtype microglia marker CD86 and its release product IL-1?.5.In the SH-SY5 Y cells culture,100 nM Rotenone inhibited cell viability of SH-SY5 Y cells to 69% of control group;while H4 R antagonist JNJ had no significant effect on the decrease of cell viability in SH-SY5 Y cells induced by rotenone.In the immunocytochemical staining and western blot assay,the expression of ?-synuclein in SH-SY5 Y cells significantly increased(169% of control group),but the H4 R antagonist JNJ did not show any significant effect on the increased expression of ?-synuclein induced by rotenone.Conclusions:Histamine H4 receptor antagonist may play a protective effect on degenerated dopaminergic neurons in PD rats induced by rotenone.The extracellular mechanism may be related to the inhibitory effect of H4 R antagonist on the proliferation and release of M1 subtypes microglia;while the direct effect of H4 R antagonist on dopaminergic neurons needs further study to clarify.This study provides a theoretical basis for the discovery of new drug targets in clinical treatment of PD,and extends the role and value of H4 R in other neurodegenerative diseases. |
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