Mechanism Of Parthenolide In Promoting Nestin-Expressing Progenitors To Repair The Developing Cerebellum After Irradiation

Background:Parthenolide（PTL）,a sesquiterpene lactone extracted from the flower buds of Tanacetum in Compositae,is commonly applied in the treatment of migraine,as well as arthritis,rheumatism,dysmenorrhea,leukemia,etc.It has analgesic,anti-tumor,anti-fungal,anti-bacterial,and migraine pharmacological effects.Feverfew is a perennial herb with a height of 60cm.Leaves alternate and pinnate.The head flower is on the top of the branch,and the tongue flower is white.Native to Southeast Europe,Feverfew is widely grown in Europe,North America,and Australia.Seed or cutting propagation,like good drainage,sunny areas.At present,the crude plants containing PTL include Compositae,Magnoliaceae,Magnolia,and Michelia.According to traditional Chinese medicine,these plants can clear away heat,detoxify,expel wind and dehumidify.Recent studies have shown that PTL has a variety of significant pharmacological activities.For peripheral nerve injury,PTL promoted sciatic nerve regeneration by regulating the activity of microtubule tyrosinase.As a small molecule drug,PTL could enter the blood-brain barrier and penetrate the central nervous system.More and more research revealed that PTL plays a neuroprotective role in cerebral hemorrhage and ischemia models.However,most of these studies concentrated on differentiated neural cells.The effect of PTL on neural stem cells and neural progenitor cells with proliferation ability remains unclear.The cerebellum is only 10%size of the human brain,but it contains most of the mature neurons in the adult brain.Cerebellar growth occurs mainly after birth in mice and is due to rapid proliferation in external granule layer（EGL）.Proliferation of cerebellar granule neural precursors（GNPs）in the EGL peaks at postnatal day 7（P7）and ends at P14 as the GNPs exit the cell cycle and migrate to the internal granular layer（IGL）,differentiate into cerebellar granule neurons.The cerebellum plays a major role not only in maintaining motor coordination,but also in processing perception,cognition,and emotion.Consequently,it is an excellent model for studying the development and regeneration of neurons.X-ray could cause neuron degeneration and necrosis in the central nervous system（CNS）.Once it occurs,it is irreversible and gets a poor prognosis.At present,there is no recognized effective treatment in clinical.It has proved that a few GNPs can survive,proliferate and reconstruct EGL under the action of a single X-ray dose during the early postnatal period.Recent studies have revealed that a population of cerebellar Nestin-expressing precursors（NEPs）is indispensable for regeneration of injured developing cerebellum,meanwhile,SHH is valuable for this process.SHH also regulates normal cell development,but aberrant activation of SHH signaling can cause hyperproliferation and malignancy.Nevertheless,there is a lack of reports about exogenous drugs to protect GNPs from radiation and contribute to the expansion of NEPs for regeneration.Our results indicate that PTL is a promising candidate drug for cerebellar regeneration and provide a theoretical basis for the new application development of related pharmacognostic plants,which has far-reaching significance.Purposes:1.To study the protective effect of PTL on GNPs in EGL of the developing cerebellum after irradiation2.To explore the effect of PTL on NEPs in the process of EGL repair after irradiation3.To elucidate the molecular mechanism of PTL promoting the repair of the developing cerebellum after irradiationMethodsX-RAD RS2000 instrument used for radiation damage;Primary culture of precursor cells of granule neurons:the cerebellar tissues digested with papain,extracted by Percoll gradient centrifugation,and cultured on coated cover slides with Neurobasal/B-27 culture system;In neural stem cells:for self-renewal,we mainly used neural stem cells conditioned medium supplemented with EGF and b FGF to compare their ability to produce neurospheres in vitro.For pluripotent differentiation,we used different differentiation culture conditions and immunofluorescence staining to determine the ability to differentiate into granular neurons（PAX6 as the marker）,astrocytes（S100β,GFAP as the marker）,and Purkinje cells（Calbindin as the marker）;Lineage tracing assay used to determine the fate of NEPs:after tamoxifen treatment,Nestin-cre ER^T2/Rosa GFP have activated the expression of GFP in the cells,and all cells that had expressed Nestin labeled with GFP;Immunofluorescence staining:the expression of apoptosis,DNA damage,proliferation,and other related markers observed in frozen sections or cell climbing slices;Western Blot used to detect the expression level of target protein in cell or tissue samples;Real-time fluorescence quantitative PCR used to detect m RNA expression of different target genes in cells or tissues.ResultsThis study includes three parts:Part One:To explore the protective effect of PTL on acute radiation injury of the developing cerebellum1.The effect of PTL on GNPsMedulloblastoma（MB）model animals:Ptch lac z^+/-and Math1 cre/Ptch^flox/flox transgenic mice were used respectively.It suggested that PTL had no obvious cytotoxicity to MB cells and GNPs,even ptomoted the proliferation of GNPs in vivo.Combined with in vitro cytotoxicity experiment,it revealed that PTL may regulate the cerebellar microenvironment.2.The protective effect of PTL pretreatment on GNPs in EGL of the developing cerebellum after irradiation（IR）The cerebellum of P4 mice was irradiated with a single dose of 4Gy X-ray.Immunofluorescence staining of frozen sections showed that compared with the Non IR group,the apoptosis marker cleaved-caspase3 and DNA damage markerγ-H2AX in EGL significantly decreased after treated with PTL before irradiation,indicating that PTL reduced the damage of GNPs after irradiation.PTL inhibits the NF-κB signaling pathway,and BAY11-7802 has the same inhibitory target as PTL,so it is used as a reference product.In vitro,inhibition of NF-κB activation was further confirmed in PC-12 cell lines.It indicated that PTL might partly reduce the damage of GNPs in the developing cerebellum by inhibiting the activation of NF-κB.However,when BAY11-7802 was injected in vivo,P4 mice could not survive for more than 24 hours,and the proliferation of GNPs in EGL could only observe in PTL IR mice.No similar results were observed in BAY 11-7802 IR mice.Recent study emphasized that a single low-dose of PTL significantly promoted regeneration of injured sciatic nerve axonal by interfering with detyrosination of alpha-tubulin in an NF-κB-independent manner.These results suggest that PTL can play a neuroprotective role through other effects.ROS induced by radiation is one of the critical causes of radiation injury.PTL can regulate the redox state in cells.In vivo,PTL inhibited H₂O₂ production in the cerebellum after IR and promoted glutathione reductase and glutathione synthetase production.In vitro,it also confirmed that PTL inhibited ROS production in PC-12,suggesting that the protective effect of PTL on acute radiation injury may be related to the reduction of ROS levels in the cerebellum.Part Two:To explore the effect of PTL on NEPs in the process of EGL repair in the developing cerebellum after irradiation1.PTL promoted the repair of EGL after radiation injury in the developing cerebellumThe cerebellum of mice continued to develop postnatally until 21 days after birth（P21）.The decisive periods for observing cerebellar development were P7-P8,P14,P21,and P30.We found the effect of a single dose of PTL on the development of the mouse cerebellum at several time points by immunofluorescence staining of frozen sections.The results showed that PTL promoted the proliferation of GNPs in EGL of the cerebellum after IR at P8 and P14.Previous studies have confirmed that although the developing cerebellum can complete self-repair after IR,the volume of repaired cerebellum after maturation was significantly smaller than that of the normal cerebellum.In observation of the mature cerebellum（P30）,the sagittal section area of the cerebellum after PTL pretreatment was remarkably sizable than that of IR alone.By immunofluorescence staining at P8,P14,and P21,we detected the corresponding neural cell markers.With PTL treatment,the number of neural stem cells,astrocytes,and granule precursors increased markedly.It generally believes that neural stem cells are a crucial source of neural repair.In vitro experiments,we confirmed that PTL promoted the self-renewal and differentiation of neural stem cells into neurons.In conclusion,PTL may contribute to the regeneration of GNPs in EGL after radiation injury.2.PTL affected the amplification and distribution of NEPs in EGL and PCL of the cerebellumSince some studies have confirmed that NEPs play a pivotal role in the development of cerebellar injury repair after irradiation,in this part,we mainly elucidate the relationship between PTL and NEPs.We determined the effect of PTL on the proliferation and distribution of NEPs in the developing cerebellum by using Nestin CFP fluorescent tracer transgene mice.By immunofluorescence staining,we found that PTL could promote the number of NEPs in EGL and PCL after IR at 24 hours.At P8 and P14,our results indicated that PTL increased the expression of NEPs in the cerebellum after irradiation injury and contributed to the recovery of damaged EGL.3.PTL promoted the reprogramming of NEPs in EGL and PCL to repair damaged cerebellumSome studies have pointed out that NEPs can only differentiate into GABAergic intermediate neurons（such as astrocytes）under normal conditions,and could not transform glutamatergic neurons（granular neurons）.Radiation damage can reprogram NEPs and have the ability to differentiate into GNPs and mature granular neurons.In this part,we mainly determined whether PTL could promote the reprogramming of NEPs and enhance their ability to differentiate into GNPs.Using double immunofluorescence staining,we confirmed that pre-treated with PTL markedly increased the number of GFP⁺/PAX6⁺double-positive cells at each critical time point（P8,P14）,which indicated that PTL could promote the differentiation of NEPs into GNPs.On the other hand,PTL also increased the number of GFP⁺/S100β⁺double-positive cells in PCL after IR,which indicated that PTL could also increase the differentiation of intermediate neurons and keep cells in balance.Since differentiation of NEPs would lead to the GNPs loss of Nestin and SOX2 expression,confirmed the proportion of NEPs derived cells in the composition of mature cerebellar granule neurons after irradiation.We used Nestin-cre ER^T2/Rosa GFP transgenic mice and further confirmed that PTL can promote the regeneration of the cerebellum after radiation injury by promoting the reprogramming of NEPs cells.Besides,we found that PTL could directly regulate astrocytes and Purkinje cells in the cerebellar microenvironment,promoted the numbers of astrocytes,and increased the dendritic branches of Purkinje cells.Meanwhile,PTL promoted the expression of PAX2,a cell marker of intermediate neurons.These results suggested that PTL not only acted on NEPs to repair damaged cerebellum but also regulated cells in the cerebellar microenvironment to help repair the damaged cerebellum.In the cerebellum,PAX6 is one of the markers of GNPs,and GNPs are excitatory neurons in the cerebellum;PAX2 is a marker of interneurons,which are mainly inhibitory neurons in the central nervous system.These results suggested that PTL may regulate the balance of excitatory/inhibitory nerve conduction in the cerebellum after IR,which is closely related to the SHH signal.Part Three:The mechanism of PTL on sonic hedgehog（SHH）signal1.PTL promoted the secretion and expression of SHH in the cerebellum after irradiationImmunofluorescence staining EDU/Tuj1 in regular primary GNPs showed that PTL did not affect the proliferation and differentiation of GNPs in sonic hedgehog conditioned medium（SHH-CM）.Western blotting of the cerebellar tissue samples confirmed that PTL could promote the expression of SHH in the cerebellum after irradiation.QRT-PCR results showed that PTL promoted SHH m RNA expression in the cerebellum after irradiation.At the same time,we found that PTL promoted the expression of Dispatched-1 in the cerebellum at the m RNA level after irradiation.The immunofluorescence staining in the frozen sections showed that PTL increased the dendritic density and secondary branches in PCs after irradiation,which suggested that PTL might promote more secretion of SHH in PCs to act on GNPs in EGL.At the same time,we found that PTL increased the expression of S100β,a marker of astrocytes.It could increase SHH secretion because of more astrocytes in the cerebellum.The frozen sections were stained with Calbindin/SHH or S100β/SHH.The results showed that PTL promoted the expression of SHH in PCs and astrocytes.These results suggested that PTL could promote SHH secretion in the cerebellum after irradiation.2.PTL promoted the secretion of SHH in astrocytes by inhibiting the PI3K/AKT pathwayIt has been reported that in the process of the cerebellum after radiation damage,irradiation had more effect on GNPs and astrocytes but little effect on Purkinje cells.We had previously examined the effect of PTL on GNPs after IR in the cerebellum.Therefore,we explored the outcome of PTL on astrocytes.Astrocytes support and regulate the function of neurons.After the injury of the central nervous system by exogenous chemicals or trauma,astrocytes in the injured area can form a glial"Scar".It has been shown that astrocytes are one of the sources of SHH in the cerebellar microenvironment under pathological conditions.In primary astrocytes,immunofluorescence staining results showed that PTL could significantly promote SHH expression.QRT-PCR and Western Blot results showed that PTL promoted SHH expression in primary astrocytes.In the astrocyte cell line C8-D1A cells,we found that PTL had a significant time-dependent inhibitory effect on the PI3K/AKT/GSK3 pathway.Meanwhile,Western Blot results showed that in C8-D1A cells,AKT inhibitor Triciribine and PI3K inhibitor LY 294002promoted SHH expression,and AKT activator SC-79 inhibited SHH expression.But PTL could reverse the inhibition of SC-79 on SHH.Western Blot results showed that PTL inhibited the expression of BAX and cleaved-caspase3,and promoted the expression of BCL2.After pretreatment with PTL combined with SC-79 and Triciribine,PTL increased the expression of SHH in the cerebellum after IR by inhibiting the AKT pathway.In conclusion,PTL can promote SHH expression in astrocytes by inhibiting the AKT pathway,which can reduce the radiation damage in C8-D1A.PTL also promoted SHH expression in the cerebellum by inhibiting the AKT pathway.Conclusion1.In the concentration of PTL used in this study,there is no serious cytotoxicity to GNPs in vitro,but it can maintain the proliferation of GNPs to a certain extent in vivo.On the one hand,it protects GNPs from apoptosis caused by acute radiation injury by inhibiting ROS;on the other hand,it promotes the repair and regeneration of damaged EGL.2.PTL pretreatment can promote the expansion and reprogramming differentiation of NEPs in EGL and PCL of the developing cerebellum,it is crucial for repair after IR damage.PTL promotes the recovery of excitatory GNPs in EGL and the number of inhibitory interneurons.All in all,it maintains the overall balance of the cerebellar nerve cells.3.PTL regulates the expression and release of SHH in the cerebellar microenvironment through PI3K/AKT pathway and promotes the amplification and reprogramming of NEPs through the role of SHH,thus completing the repair of the irradiated cerebellum.