Ginkgolide B's Neuroprotective Effect On CPZ-induced Demyelination And MPTP-PD Mice And Its Mechanism Based On The Theory Of Different Diseases With The Same Treatment

Multiple sclerosis(MS)is an inflammatory demyelinating disease of the central nervous system(CNS).The main pathological features are the infiltration of inflammatory cells,the mutation of axonal,and eventually severe demyelination.Cuprizone(CPZ)-induced demyelinating model is a classic model for studying MS.Parkinson's disease(PD)is a degenerative disease in the CNS,in which the main pathological manifestation is the degeneration of dopaminergic(DA)neuron in substantia nigra pars compacta(SNpc).The mouse model induced by1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)is the classic model to study PD.A large number of clinical and animal experiments have proved that inflammatory responses play an important role in the development of MS and PD,in which the inflammatory cells enter the CNS through the impaired blood-brain barrier.Microglia and astrocytes are effector cells of neuroinflammation.After CNS damage,glial cells are rapidly activated,and then inflammatory factors are released,so immune responses and tissue repair are initiating.Once repair is complete,the glial cells return to a resting state.In neuroinflammatory degenerative diseases,glial cells are activated repeatedly,and persistently active signals can induce chronic neuroinflammatory responses.The pathogenesis of MS and PD is not clear.However,there is no doubt that glial cell-mediated inflammation is involved in the development of MS and PD.Ginkgolide B(GB)is a terpene lactone in Ginkgo biloba extract.It is widely used in the treatment of neurological diseases including cerebral ischemia.Some experiments showed that GB can inhibit inflammatory response.Basing on the concept of different diseases with the same treatment,this study clarifies the therapeutic effects and the same mechanism of GB in MS and PD disease models.Part ? Protective effect of Ginkgolide B on CPZ-induced demyelinating mice and its mechanismObjectives:In this study,CPZ-induced C57BL/6 mice were used to establish a CNS demyelinating model and then were given GB by intraperitoneal injection.We observe the behavioral performance,and investigate the molecular mechanism about astrocytes,microglia and oligodendrocytes,in order to clarify the neuroprotective effect of GB on CNS demyelination.Our aim is to provide laboratory evidence for the future application of GB in the treatment of MS.Methods:The study consists of in vivo and in vitro experiments.Six-week-old C57BL/6 male mice were used for in vivo experiments to establish a CPZ-induced demyelinating model.Mice were divided into normal group,CPZ group,CPZ+GB group,8 mice in each group.Normal mice were fed with conventional feed for 6weeks.CPZ group and CPZ+GB group were fed with 0.2%CPZ-containing feed for 6weeks.At the beginning of the fourth weekend(day 28),the GB+CPZ group were received GB by intraperitoneal injection once a day and continued to the sixth weekend(day 42).At the same time,the normal group and CPZ group were given saline by intraperitoneally injection every day.The weight was recorded from the beginning of modeling.The behavioral test was performed during the administration.At the end of the sixth week,brain tissue was taken.Four mice of each group were used for tissue sections.Then Luxol Fast Blue(LFB),Black Gold II and MBP staining were performed to observe the morphology and loss of myelin sheath.The changes of glial cells were observed by immunofluorescent staining.The brain proteins were raised in remaining four mice of each group,,and the expression of inflammatory factors in brain was detected by ELISA.The in vitro cell experiment was conducted in culture primary astrocytes,BV2 and oligodendrocyte PDGF R?at the condition of 37°C and 5%CO2.The three kinds of cells were set to PBS control group,25?g/ml and 50?g/ml GB group.In the GB group,immunocytochemical staining was performed to observe the expression of inflammatory factors and trophic factors on the cells.The experimental data were analyzed by GraphPad Prism 5.0statistical software.Result:1.In the first week of CPZ feeding,compared with the normal group,the weight of the mice in the CPZ group was significantly reduced,and keeping stable and low for the next three weeks.In the following two weeks,compared with the CPZ group,mice in the CPZ+GB group generally gained weight,and their behavioral performance improved significantly.In the forced swimming test,the cumulative distance of swimming in the CPZ group was less than that in the CPZ+GB group(p<0.05).The rest time of swimming in the CPZ group was longer than that in the CPZ+GB group(p<0.01).In the elevated cross-maze test,the entry times into the open arms in CPZ group was less than the CPZ+GB group(p<0.05).In the T-maze test,the entry times into the food area in CPZ group was less than the CPZ+GB group(p<0.05).2.The LFB staining,black gold staining,and MBP staining in corpus callosum showed severe myelin loss in the CPZ group,and the myelin loss was significantly reduced in the CPZ+GB group comparing with the CPZ group(p<0.001,p<0.0001,respectively).3.The expression of Iba-1 in CPZ+GB group was significantly inhibited comparing with the CPZ group(p<0.001).The immunofluorescence results showed that compared with the CPZ group,GB treatment significantly inhibited the expression of Iba1+TLR4+and Iba1+NF-?B+(p<0.01,p<0.001,respectively).Western blot results showed that compared with the CPZ group,the expressions of TLR4 and NF-?B in the CPZ+GB group decreased(p<0.05,p<0.001,respectively).The results showed that compared with the CPZ group,the CPZ+GB group significantly inhibited the expression of IL-1?and TNF-?when detected with ELISA(p<0.01,p<0.001,respectively).4.Compared with the CPZ group,the expression of Iba1~+Arg-1~+was increased in the CPZ+GB group(p<0.001),and the expression of Iba1~+iNOS~+was decreased(p<0.01)when detected by immunofluorescence double staining.Western blot results showed that the expression of Arg-1 in the CPZ group was reduced(p<0.01),and the expression of iNOS in the CPZ group was higher than the CPZ+GB group(p<0.01).5.With regard to BV2 stimulated by LPS,compared with the PBS control group,the 50?g/ml GB group reduced the expression of NF-?B and iNOS,and increased the expression of Arg-1(p<0.01,p<0.001,respectively).6.Compared with the CPZ group,the expression of GFAP in the CPZ+GB group was reduced in the corpus callosum,hippocampus,and cortex(p<0.01)using immunohistochemistry and western blot.Immunohistochemical results showed that compared with the CPZ group,GB treatment effectively induced the expression of BDNF on astrocytes(p<0.05,p<0.01,respectively).Similarly,the CPZ group can slightly induce the expression of GDNF on astrocytes(p<0.01).After GB treatment,the expression of GDNF on astrocytes was up-regulated in the corpus callosum and subventricular area(SVZ)(p<0.001).7.The immunocytochemistrical results showed that compared with the PBS control group,the GB group(25?g/ml and 50?g/ml,respectively)increased the expression of GDNF and BDNF(p<0.01,p<0.001,respectively)when the primary astrocytes were stimulated by LPS.The PCR results were consistent with the results of immunocytochemistry.8.The NG2~+was rapidly generated in the corpus callosum and SVZ regions in demyelinated mice,and the CPZ+GB group further promoted NG2~+production in these regions when immunofluorescence double staining was used.Compared with the CPZ group,the CPZ+GB group increased the expression of Ki67 significantly on NG2~+(p<0.001),indicating that GB-induced NG2 was proliferating.9.The immunocytochemistrical results showed that compared with the PBS control group,low concentration of GB(25?g/ml)slightly induced PDGFR?~+expression(p<0.05),while high concentration of GB(50?g/ml)induced PDGFR?high expression significantly(p<0.001).Conclusion:1.GB has a significant protective effect on CPZ-induced demyelinating injury,and the mice with GB treatment show significant improvements in behavior and pathology.2.GB inhibits the expression of inflammatory factors related to microglia,and promotes the expression of neurotrophic factors on astrocytes.GB may participate in the repair and regeneration of myelin sheaths by dynamically adjusting the balance of microglia and astrocytesPart 2 Protective effect of Ginkgolide B on MPTP-induced Parkinson mice and its mechanismObjective:In this study,MPTP-induced C57BL/6 mice were used to establish a PD model,then mice were given GB by intraperitoneal inject.The gait of mice and the repair of dopaminergic neurons in substantia nigra were observed after administration.We investigated the mechanism of microglia and astrocyte in order to clarify the neuroprotective effect of GB in PD,and further provide a scientific theoretical basis for GB to treat PD in the future..Method:This study consists of experiments both in vitro and in vivo.Human dopaminergic neuroblastoma SH-SY5Y cells were treated with MPP~+to establish a PD cell model,and then GB at different concentrations(12.5?g/ml,25?g/ml,and 50?g/ml)were used to intervene cells.These cells are divided into PBS control group,MPP~+model group,12.5?g/ml GB group,25?g/ml GB group,and 50?g/ml GB group.Immunocytochemistry was used to detect the expression of tyrosine hydroxylase(TH),and the slides were blindly observed under a confocal microscope.C57BL/6 male mice were used to establish MPTP-induced PD models.Mice were divided into normal group,PD group and PD+GB group,8 mice in each group.Mice in the control group were fed with conventional feed for 2 weeks.In the first week,PD group and PD+GB group were intraperitoneally injected with MPTP(15 mg/kg on the first day,20 mg/kg on the second day,and 30mg/kg on the 3-7 days).In the second week,the mice in PD group was intraperitoneally injected with saline,and the mice in PD+GB group was intraperitoneally injected with GB(20 mg/kg)once a day.Gait analysis was performed during the administration period.The brains were taken at the end of the 15th day,and divided into two parts:protein extraction and tissue sections.The dopaminogic neurons and changes on microglia and astrocytes were observed by TH staining and immunofluorescence staining.The experimental data were analyzed by GraphPad Prism 5.0 statistical software.Result:1.The immunocytochemical results showed that compared with the MPP~+model group,a high concentration of GB(50?g/ml)could significantly promote the SH-SY5Y induced by MPP~+expresses TH(p<0.001).2.The immunohistochemical results showed that compared with the PD group,the GB increased the expression of TH~+(p<0.001).Western blot showed that compared with the PD group,the expression of TH in the PD+GB group was increased(p<0.05).3.The immunohistochemical and Western blot results showed that compared with the PD model group,the expression of Iba1~+in the PD+GB group was reduced(p<0.001,p<0.001 respectively).The immunofluorescence double staining showed that compared with the PD group,the expression of Iba1~+iNOS~+in the PD+GB group was reduced(p<0.001),and there was no significant difference in the expression of Iba1~+Arg-1~+between PD group and PD+GB group.4.Immunohistochemical staining showed that compared with the PD group,the expression of GFAP~+in the PD+GB group was slightly,but significantly lower(p<0.05).5.Immunofluorescence double staining showed that compared with the PD group,the expression of Iba1~+TLR4~+in the PD+GB group was reduced(p<0.001).Similarly,compared with the PD group,the expression of GFAP~+TLR4~+in the PD+GB group was reduced(p<0.05).ELISA results showed that compared with the PD group,GB inhibited the expression of TNF-?(p<0.05).Conclusion:1.GB has a significant protective effect on the damaged DA neurons in substantia nigra,and significant improvements in behavior and pathology were observed in the treated mice.2.GB can inhibit the expression of inflammatory factors related to microglia and astrocytes,and play a role in neuroprotection by inhibiting the inflammatory talk of microglia and astrocytes.