| ObjectiveTo study whether Optimized Yinxieling Formula(OYF)can play a neuroprotective role in 6-OHDA-induced PD rat model and MPTP-induced PD mouse model through anti-inflammatory effect.To study the anti-inflammatory effects of serum containing OYF on activation of microglial BV2 cells induced by lipopolysaccharide(LPS)and Raw 264.7 macrophage cells induced by IFN?.To study the influence of OYF on gene expression in PD animal model midbrain tissue and to explore the molecular mechanism and drug targets of its neuroprotective effect by using transcriptome sequencing technology.MethodsThirty-six SD rats were randomly divided into sham group,model group and OYF group(12.9 g·kg-1).The PD rat models were established by stereotaxic microinjection of 6-OHDA into one side of striatum.Apply OYF 7 days before making model,one time each day.OYF was administrated orally through intragastric tube for eight weeks after the operation were successfully performed.Apomorphine(APO)-induced rotation test and step adjustment test were used to determine the behavioral changes of rats in each group.The morphological and quantitative changes of DA neurons(TH-positive)and microglia cells(Iba-1-positive)in midbrain subtantia nigra of three groups were examined by immunohistochemical method.The expression of TH,COX-2 and iNOS at the protein level were detected by Western blot.And the expressions of cyclooxygenase-2(COX-2),iNOS,tumor necrosis factor-?(TNF-?),IL-1?and IL-6 at the mRNA level were detected by RT-PCR.Thirty-six male C57BL/6 mice were randomly divided into control group,model group and OYF(25.8 g·kg-1)group.PD model was established by continuous intraperitoneal injection of MPTP(18 mg·kg-1)for 4 times every 2 h.The treatment group was given OYF by intragastric administration before and after modeling for successive 14 days(7 days for each).Pole and rotarod tests were used to observe neuroethology of mice.The morphological and quantitative changes of DA neurons(TH-positive)and microglia cells(Iba-1-positive)in midbrain subtantia nigra of three groups were assessed by immunohistochemical method.TH,COX-2 and iNOS protein expressions were detected by Western blot.And the expressions of COX-2,iNOS,TNF-?,IL-1?and IL-6 at the mRNA level were detected by RT-PCR.In vitro inflammatory cell models were established on LPS stimulated BV2 microglial cells and IFN-? induced RAW264.7 macrophage cells.After pretreatment with different concentration of serum containing OYF,cell relative viability,released content of NO and the mRNA expression of inflammation associated proteins(COX-2 and iNOS)and inflammatory factors(TNF-?,IL-1? and IL-6)were detected by the MTT,Griess assay and RT-PCR,respectively.After PD model rats or mice treated with OYF for 9 weeks or 14 days respectively,midbrain was separated to extract total RNA by Trizol method.And the quantification and quality of obtained RNA were detected by agarose gel electrophoresis and Agilent 2100 Bioanalyzer.High-throughput transcription sequencing(RNA-seq)was used to scan differentially expressed genes.GO and KEGG were used to analyze their bioinformatics.Trend analysis was performed on ShortTime-series Expression Miner.Expression files of differential genes were input and parameter(-pro 20-ratio 1.0)was selected(Samples were sorted by biological logic).Functional enrichment analysis of each trend was performed on GO or KEGG.P value was obtained by hypothesis testing and corrected by FDR.Q value?0.05 as threshold,GO term and Pathway satisfying the condition were defined as significant enrichment of GO term and Pathway in the trend.ResultsNeuroethology:After APO inducing,rats in sham group exhibited normal activities and showed no abnormal rotational behaviors.Compared with model group,rotations of rats in OYF group on week 2,4,6 and 8 after operation were all obviously decreased(P<0.05 or P<0.01).The number of TH-positive neurons in lesioned side midbrain subtantia nigra of model group was significantly decreased,compared with those of the sham group(P<0.01),meanwhile the number of microglia cells(Iba-1-positive)was significantly increased(P<0.05)with amoeba-like shape,and the mRNA and protein expression of COX-2 and iNOS were significantly increased(P<0.05).Compared with model group,the number of TH-positive neurons in lesioned side midbrain substantia nigra of OYF group significantly increased(P<0.05),meanwhile the number of Iba-1-positive cells was significantly decreased(P<0.05),and the expression of COX-2 and iNOS mRNA at both mRNA and protein level were significantly decreased(.P<0.05).The results of RT-PCR showed that,the TH mRNA expression of model group was obviously decreased compared with those of the sham group,while TH mRNA expression of OYF group was significantly increased compared with model group(P<0.05).The expression of TNF-? and IL-1? at mRNA level were obviously increased compared with sham group,while TNF-? mRNA and IL-1? mRNA in control group were obviously decreased compared with model group.Neuroethology:Compared with control group,the time of climbing pole was significantly prolonged and the time mice on the rotarod was decreased significantly in the model group.Compared with model group,the time of climbing pole was significantly shortened and the time mice on the rotarod was significantly increased in OYF group.Compared with control group,the number of DA neurons(TH-positive)in midbrain subtantia nigra significantly decreased(P<0.01),while the number of microglia cells(Iba-lpositive)significantly increased(P<0.05)with amoeba-like shape,the mRNA and protein expression of COX-2 and iNOS were significantly increased(P<0.05)in the model group.Compared with model group,the number of DA neurons(TH-positive)in midbrain subtantia nigra significantly increased(P<0.05),while the number of microglia cells(Iba-lpositive)significantly decreased(P<0.05)with amoeba-like shape,the mRNA and protein expression of COX-2 and iNOS were significantly decreased(P<0.05)in the OYF group.The results of RT-PCR showed that,compared with control group the TH mRNA expression of model group was obviously decreased,while TH mRNA expression of OYF group was significantly increased compared with model group(P<0.05).The expression of TNF-? and IL-1? mRNA was obviously increased compared with control group,while their mRNA expression in OYF group were significantly decreased compared with model group.The results of MTT showed after treated with different concentration of serum containing OYF and 100 ng·mL-1 LPS or 50 U·mL-1 IFN-? on BV2 or RAW 264.7 for 24h,cell survival rates had no statistical significance compared with control group,which indicated that in the experimental dose range different concentration of serum containing OYF had no cytotoxicity on activated BV2 or RAW 264.7 cell.Compared with control group,treated with LPS or IFN-?evidently increased the secretion level of inflammatory mediator NO from BV2 or RAW 264.7 cells.NO release of activated BV2 or RAW 264.7 cells could be inhibited by pretreatment with different concentration of serum containing OYF.The results of RT-PCR demonstrated that treated with LPS or IFN-y evidently increased the expression of inflammatory proteins(COX-2 and iNOS)and inflammatory factors(TNF-? and IL-1?)of BV2 or RAW 264.7 cells.While pretreatment with different concentration of serum containing OYF could inhibit inflammatory proteins(COX-2 and iNOS)and inflammatory factors(TNF-? and IL-1?)expression of activated BV2 or RAW 264.7 cells.118 genes evidently increased and 277 decreased in midbrain of PD model group compared with sham group.398 genes obviously increased and 30 decreased in OYF group compared with model group.Through GO and KO analysis on differentially expressed genes,OYF could exert its protective function on damaged cells by regulating neurotransmitters metabolism,cell growth and death,signal transduction,cell communication,stress response and neurotransmitters metabolism.118 genes evidently increased and 277 decreased in midbrain of PD model group compared with sham group.398 genes obviously increased and 30 decreased in OYF group compared with model group.Through GO and KO analysis on differentially expressed genes,OYF could exert its protective function on damaged cells by regulating neurotransmitters metabolism,cell growth and death,signal transduction,cell communication,stress response and neurotransmitters metabolism.ConclusionOYF has protective effects on 6-OHDA induced PD rat DA neuron damage and can inhibit activation of microglial cells and the expression of related inflammatory factors in subtantia nigra.OYF can improve exercise performance of PD mice,has protective effects on MPTP induced DA neurons damage and can inhibit activation of microglial cells and the expression of related inflammatory factors in subtantia nigra.OYF medicated serum can obviously inhibit inflammatory response of BV2 microglial cells and RAW264.7 macrophage cells in vitro.The results of transcriptome sequencing suggest that the main mechanism of OYP in treating PD is related to amino acid and lipid metabolism improvement,and suppression of oxidative stress injury and neovascularization. |
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