Therapeutic Effect Of Nasal Administration Of Controlled-Release Icariin Temperature-Sensitive Gel On Mouse Model Of Parkinson’s Disease

Parkinson’s disease(PD),is also known as tremor paralysis,is a chronic degenerative disease of the central nervous system.As a disease whose prevalence is related with advancing age,it is the second-largest degenerative disease in humans after Alzheimer’s disease.It is characterized by substantia nigra progressive degeneration of dopaminergic neurons in the compact,leading to persistent cognitive and behavioral disorders.PD is a kind of hereditary and sporadic disease,which is related to genetic and environmental factors.However,the causes and the pathogenic mechanism of PD remain unknown.At present,levodopa(L-DOPA)is still the mainstream drug for the clinical treatment of PD.It is mainly to improve the symptoms by supplementing the dopamine,but it cannot delay the progress of the disease.Taking these drugs,particularly over a long stretch of time,can cause a decreasing in the efficacy,at the same time,it can also lead to serious side effects when taken long-term.Icariin(ICAR)is a flavonoid compound extracted from the traditional Chinese medicine Epimedium.It is also one of the main active ingredients for treating PD in traditional Chinese medicine.It has been shown to have neuroprotective effects and can protect the dopaminergic neurons in PD animal models.But the clinical application has been limited by poor water solubility and low oral bioavailability.According to clinical needs,a new dosage form and drug delivery systems is very important.In this study,a novel icariin/hydroxypropyl-β-cyclodextrin inclusion complex(ICAR/HP-β-CD)drug delivery system was constructed,which can improve the water solubility and the bioavailability of ICAR.It can also achieve the purpose of the controlled release of therapeutic agents.Under the temperature of the nasal cavity,ICAR/HP-β-CD can assemble into a temperature-sensitive gel(ICAR/HP-β-CD-ATG),it can reach the administration place quickly and credibly,and then slowly release ICAR,so as to achieve a method for therapeutic purposes.In the study of the first part,the inclusion method of saturated aqueous solution was used to prepare ICAR/HP-β-CD.The optimal technological parameters were obtained by the orthogonal tests.The phase solubility-curve showed the inclusion proportion and equilibrium-constant.The properties of the ICAR/HP-β-CD were characterized by fourier transform infrared spectroscopy(FT-IR)analysis,differential scanning calorimetry(DSC)analysis,~1H-NMR analysis and thin layer chromatography(TLC)analysis.The orthogonal test results show that the optimal formula for preparing ICAR/HP-β-CD was 6%(w/v)HP-β-CD,4 h,35℃,and the drug loading rate of ICAR/HP-β-CD prepared under these conditions was 7.99%.Characterization analysis results showed that ICAR could be embedded in HP-β-CD.In the study of the second part,cold magnetic stirring was used to prepare ICAR/HP-β-CD-ATG by using the reverse temperature-sensitive properties of poloxamer.The physiological temperature of the nasal cavity as an indicator,an orthogonal test was used to investigate the preparation and preparation of the optimal gelling temperature.Phosphate buffer solution with the same p H simulate the nasal cavity medium for the drug in vitro release study.The cumulative release of the drug is used to evaluate the sustained release effect of the system.In the formulation screening of ATG,gel formulations were determined based on rheological properties:poloxamer 407:poloxamer 188 at a ratio of 16:2.In vitro ICAR release profiles from ICAR/HP-β-CD-ATG micelles showed that the cumulative release rate of the gel can reach98.64±1.08%by 24 h.It shows that poloxamer can be a good release carrier for ICAR.In the study of the third part,paraquat(PQ)thermosensitive gel was administered by intranasal administration to establish a mouse model of PD.The small animal imaging technology was used to investigate the tissue distribution and metabolism of rhodamine B(RB)-labeled ICAR/HP-β-CD-ATG in normal mice.The effects of ICAR on the behavioral capacity of PD mice were investigated through open field tests,suspension tests,pole climbing tests,and swimming tests.Dopamine(DA)and its metabolites dihydroxyphenylacetic acid(DOPAC)and homovanillic acid(HVA)in the striatum of mouse brain tissue were investigated by high-performance liquid chromatography with electrochemical detection.Real-time RT-PCR was used to detect the expression of inflammatory factors,apoptotic factors,oxidative stress factors in the striatum of PD mice.A biochemical kit was used to detect the enzyme activity of mitochondrial complex I in the striatum of PD mice.The apoptotic cells in the substantia nigra were assayed using TUNEL method.These research was designed to study the therapeutic effect of ICAR/HP-β-CD-ATG nasal administration on mice with PD and its mechanism for PD.Real-time RT-PCR experimental results indicated that ICAR/HP-β-CD-ATG can reduce the inflammatory response in the striatum of mice by down-regulating the Interleukin-1β(IL-1β)and Monocyte chemoattractant protein-1(Mcp-1)expression,and inhibit the levels of apoptotic factors in the striatum of mice by up-regulating the expression of Bcl-2,Bcl-x L and down-regulating the expression of Caspase-3and Bax.Moreover,ICAR/HP-β-CD-ATG also decrease oxidative stress in PD by down-regulating the expression of CAT and HO-1,and increase the levels of nerve growth factor receptor(Ngfr),which produced a therapeutic effect on PD.Detection of mitochondrial complex I enzyme activity results shows that ICAR can relieve PD symptoms by increasing mitochondrial complex I enzyme activity.Immunofluorescence experiments shows that the drug can improve PD symptoms in mice by improving the number of TH-positive cells,while the positive control drug L-DOPA can directly increase the DA content in the brain,but cannot increase the number of TH-positive cells in substantia nigra.The results of TUNEL experiments shows that ICAR can improve Parkinson-like symptoms in mice by reducing the number of apoptotic cells in substantia nigra,and L-DOPA can increase the number of apoptosis in substantia nigra.In conclusion,ICAR/HP-β-CD-ATG as drug carrier exhibits a good brain targeting through intranasal administration.This preparation had the advantages of prolonging the nasal retention time and enhancing the bioavailability.It exerts respective advantages in the treatment of PD.