Echinacoside treat neurodegenerative illnesses: Parkinson's disease
Mar 05, 2022
Contact: emily.li@wecistanche.com
Part Ⅱ：Neuroprotective Effects And Related Mechanisms Of Echinacoside in MPTP-Induced PD Mice
The objective of this paper is going to explore the neuroprotective effect and the related mechanisms of echinacoside (ECH) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mice. We observed the mice with Parkinson's disease which was induced by MPTP and also the neurobehaviors of mice in different groups. Then, immunohistochemistry and Western blot analysis are adopted to measure the expression of tyrosine hydroxylase (TH) and α-synuclein in the substantia nigra(SN). The content of dopamine (DA) and other neurotransmitters in the brain is detected by high-performance liquid chromatography. The expression of nerve growth factors and inflammatory factors in SN in mice in each group is measured by quantitative polymerase chain reaction. Finally, the expression of oxidative stress-related parameters in each group is measured. Through this process, we have found that the pole-climbing time among mice in the moderate and high-dose ECH (echinacoside) groups is significantly reduced (P<0.01) compared with the model group, The rotarod staying time, as well as fore and hind-limb strides, shows a significant increase(P< 0.01), as does spontaneous activity(P<0.01). Moreover, the expression levels of TH, DA, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor in SN in mice show significant increases in these two groups(P<0.01). The content of superoxide dismutase, catalase, and glutathione peroxidase indicates significant increases in the low, moderate, and high-dose ECH (echinacoside) groups (P<0.01), and the content of MDA was reduced (P<0.01).In the high-dose ECH (echinacoside) group, the expression of interleukin (IL)6 and tumor necrosis factor-α is significantly reduced (P<0.01), while the expression of IL-10 shows a marked increase(P<0.01) alongside a decrease in the expression of α-synuclein (P<0.01). Thus we concluded that ECH (echinacoside) improves neurobehavioral symptoms in PD (Parkinson's disease) mice and it also significantly increases the expression of TH and DA. The neuroprotective effect potentially correlates with anti-inflammation and anti-oxidation actions, promotes the expression of nerve growth factors, and reduces the accumulation of a-synuclein.
Keywords: Parkinson's disease, MPTP, echinacoside,α-synuclein, oxidative stress, neuroprotection, BDNF, GDNF, IL-6, TNF-α, IL-10
Echinacoside: anti-Parkinson's diseases (PD)
The Effect of ECH (echinacoside) on the Protein Expression of TH and α-Synuclein in the Substantia Nigra in MPTP-Induced PD (Parkinson's disease) Mice
The protein expression of TH in the MPTP group was significantly lower than that in the normal saline group (P<0.01). The protein expressions of TH in the EM group, EH group, and the SL group were significantly higher than that in the MPTP group (P<0.01). The protein expression of TH in the EH group was significantly higher than that in EL and EM group(P<0.01). Compared with the SL group, the difference in the protein expression of TH was not statistically significant (P>0.05). The protein expression of a-synuclein in the MPTP group was significantly higher than that in the Normal saline group(P<0.01). The protein expressions of a-synuclein in the EL group and EM group were slightly lower than that in the MPTP group, but the difference was of no statistical significance (P>0.05). Compared with the MPTP group, the protein expressions of α-synuclein significantly reduced in the EH group and SL group, and the differences were statistically significant (P<0.01)(as illustrated in Figure 4).
Figure 4 The increased protein expression of TH in the substantia nigra and decreased protein expression of α-synuclein in the substantia nigra in MPTP-induced PD mice by echinacoside.**P< 0.0I, compared with the model group; "P<0.05, "P< 0.0l, compared with the high-dose echinacoside group.
ECH (echinacoside) Reduced the Oxidative Stress Injury in the Substantia Nigra in MPTP-Induced PD (Parkinson's disease) Mice
The contents of CAT, GSH-Px, and SODin the MPTP group were significantly lower than those in the Normal saline group (P<0.05). The content of CAT in EL, EM, and EH groups and SL groups was significantly higher than that in the MPTP group(P<0.01). The contents of CAT, GSH-Px, and SOD in the EH group were all higher than those in EM and EL group (P<0.01). The differences in the contents were not statistically significant when compared with those in the SL group (P>0.05)(as shown in Figure 5A-C). The content of MDA in the MPTP group was significantly higher than that in the Normal saline group (P<0.01). The content of MDA in EL, EM, EH, and SL groups decreased significantly compared with that in the MPTP group (P<0.01). The reduction of the content of MDA was more significant in the EH group than that in EL and EM group (P<0.01). There was no significant statistical significance in the EH group in the reduction of the MDA content when compared with the SL group (P>0.05)(as shown in Figure 5D).
Figure 5 The effect of echinacoside on oxidative stress in the midbrain substantia nigra in MPTP-induced PD mice. (A) CAT;(B)GSH-PX;(C)SOD;(D) MDA.*<0.05. **P <0.01, compared with the model group; P< 0.01, compared with the high-dose echinacoside group.
ECH (echinacoside) Increased the mRNA Expression of GDNF and BDNF in the Substantia Nigra in MPTP-Induced PD (Parkinson's disease) Mice
The mRNA expressions of GDNF and BDNF in the MPTP group were significantly lower than those in the Normal saline group (P<0.01). The mRNA expressions of GDNF and BDNF in EM, EH, and SL groups significantly increased compared with those in the MPTP group(P<0.01). The mRNA expression of BDNF in the EL group was also significantly higher than that in the MPTP group (P <0.01). The mRNA expressions of GDNF and BDNF in the EH group increased significantly compared with those in EL and EM group (P<0.01)(as shown in Figure 6).
Figure 6 The increased mRNA expression of GDNF and BDNF in the substantia nigra in MPTP-induced PD mice by echinacoside. (A) GDNF;(B)BDNE.*P<0.0I, compared with the model group; "P<0.0I, compared with the high-dose echinacoside group.
ECH (echinacoside) Reduced the mRNA Expression of the Inflammatory Factors in the Substantia Nigra in MPTP-Induced PD (Parkinson's disease) Mice
The mRNA expression of IL-6 in the MPTP group significantly increased compared with that in the Normal saline group (P<0.01). The mRNA expressions of IL-6 in EM, EH, and SL groups significantly decreased compared with that in the MPTP group (P< 0.01). The reduction of the mRNA expression of IL-6 in the EH group was more significant than that in EL and EM groups (P< 0.01 and P< 0.05, respectively). There was no significant statistical difference compared with the SL group (P> 0.05)(as illustrated in Figure 7A). The mRNA expression of TNF-α in the MPTP group significantly increased compared with that in the Normal saline group (P<0.01). Compared with the MPTP group, the mRNA expressions of TNF-α in the EH group and SL group significantly reduced (P<0.01). There was no statistical difference between the two groups (P>0.05)(as shown in Figure 7B). The mRNA expression of Ⅱ-10 in the MPTP group significantly decreased compared with that in the Normal saline group (P < 0.01). Compared with the MPTP group, the mRNA expressions of IL-10 in EM, EH, and SL groups significantly increased (P<0.01). There was no statistical difference among the three groups (P>0.05)(as demonstrated in Figure 7C).
Figure 7 The reduced mRNA expression of inflammatory factors in the substantia nigra in MPTP-induced PD mice by echinacoside. (A)IL-6;(B) TNF-a;(C)IL-I0.*<0.05,**P<0.0l,compared with the model group; p< 0.05,"mP< 0.0l, compared with the high-dose echinacoside group.
Discussion
The MPTP model is widely used by researchers because it can selectively damage the dopaminergic neuron pathway in SN and striatum and can simulate most of the clinical symptoms of PD (Parkinson's disease). In the present study, an MPTP-induced PD (Parkinson's disease) model was first established. The recognized neurobehavioral evaluation methods, such as the pole test, the ability to engage in spontaneous activity, gait analysis, rotarod test, and other methods were adopted to conduct an in-depth evaluation of mice's neurobehavioral functioning. Compared with the MPTP group, mice in EM and EH groups exhibited significantly shorter pole-climbing time, significantly longer roller-residence time, and significantly longer fore and hind-limb strides. The spontaneous movement ability in mice also increased significantly. Accordingly, we believe that ECH (echinacoside) can potentially significantly improve neurobehavioral functioning in PD (Parkinson's disease) mice.
Since TH is a unique enzyme of the dopaminergic neurons and the rate-limiting enzyme in DA synthesis, the expression of DA directly reflects the content of DA in the brain.15 The results of the present study confirmed that the expression of TH in SN in both EM and EH groups increased compared with the MPTP group; furthermore, a significant increase in the content of DA and DOPAC was observed in these two groups. Considering that the abnormal aggregation of α-synuclein is the core pathological mechanism in PD (Parkinson's disease), immunohistochemistry and Western blot analysis were adopted to analyze the expression of α-synuclein, which was reduced in the EH group.
In neurodegenerative diseases, dopaminergic neurons are more susceptible to injury due to oxidative stress,'6 and oxidative stress can also lead to mitochondrial dysfunction and the accumulation of abnormal proteins. The latter two mechanisms are common causes of PD (Parkinson's disease).17 Exposure to MPTP can cause a decline in the functioning of the endogenous antioxidant system, thereby increasing the sensitivity of the dopaminergic neurons to oxidative stress. The detection of MPTP-induced oxi-dative stress can be achieved by measuring SOD, CAT, GSH-Px, MDA, and other indicators. In a cell, when the superoxide is significantly increased, SOD will increase.18 Catalase is a specific enzyme that can detoxify reactive oxygen species(ROS)and remove H, O,19 When both SOD and CAT increase, the body will have a degree of defense against the release of free radicals following an injury to the mitochondrial respiratory chain. Glutathione peroxidase is the main free radical scavenger in the human body. The increased expression of GSH-Px in the PD model has a significant protective effect on injured dopaminergic neurons. Maintaining GSH-Px viability under oxidative stress is crucial for cell survival.20As a metabolite of lipid peroxidation reaction between oxygen free radicals and unsaturated fatty acids in biomembranes, changes in MDA content can reflect the degree of lipid peroxidation in tissue, as well as changes in the content of oxygen free radicals.21 Malondialdehyde is widely used in cell and animal experiments to evaluate lipid peroxidation.22 Studies have confirmed that ECH (echinacoside) has significant free radical scavenging activity and anti-lipid peroxidation effects.
It was found that several phenolic hydroxyl groups in the molecule positively correlated with the antioxidant capacity.3.24 According to recent research reports, ECH (echinacoside) significantly reduces the generation of ROS induced by MPPt, thereby increasing cell survival. The present study found that the contents of sOD, CAT, and GSH-Px in the MPTP group were significantly lower than those in the normal saline group; however, the MDA content was significantly higher than that in the normal saline group. The content of antioxidants SOD, CAT, and GSH-Px significantly increased in EL, EM, and EH groups, while the MDA content was significantly reduced. This was consistent with results reported in the above literature, confirming that ECH (echinacoside) may have a significant effect on anti-oxidative stress in MPTP-induced PD (Parkinson's disease) mice.
It is known that GDNF and BDNF are the two most important neurotrophic factors for treating degenerative diseases of the nervous system, particularly PD(Parkinson's disease) (25). Although the effectiveness of neurotrophic factors in the treatment of PD (Parkinson's disease) has been confirmed in cell and animal models, it is difficult for the neurotrophic factor to pass through the blood-brain barrier because it is a macromolecular peptide. Moreover, because of several issues, such as infection, immune response, and unstable efficacy, in the administration modes of these drugs, their widespread application cannot be extended to clinical practice.26 Therefore, the development/search for a drug that can express or increase endogenous neurotrophic factors may have an encouraging effect on improving PD (Parkinson's disease) symptoms and even delay the progression of the disease. The present study found that the expression of GDNF and BDNF significantly increased in both EM and the EH groups. This was consistent with the results of the expression of TH in SN, and of DA in the striatum among the two groups. It was also suggested that high expression of neurotrophic factors promoted the survival of dopaminergic neurons in SN and stria-tum. The experimental results of the present study were consistent with existing experimental results conducted by our research group,692728 thereby further indicating that the neuroprotective effect of ECH (echinacoside) may be closely correlated with the promotion of the high expression of neurotrophic factors.
In the present study, the common pro-inflammatory factors IL-6, TNF-α, and anti-inflammatory factor IL-10 in SN in mice were measured by Q-PCR in all groups. The results showed that the expression of IL-6 and TNF-α were significantly reduced in the EH group, together with an increase in the expression of IL-10, thus confirming that ECH (echinacoside) may have an anti-inflammatory effect. However, there were also limitations to the current study. We did not correlate the damage generally observed in the striatum with most of the proteins, and although we performed high-performance liquid chromatography for the striatum we did not adopt a similar approach for elements in the rest of the research.
echinacoside treat PD (Parkinson's disease)
Conclusion
In summary, the present study confirmed that oxidative stress, inflammation, and abnormal protein aggregation were involved in the pathogenesis of MPTP-induced PD (Parkinson's disease) models. Echinacoside may not only improve neurobehavioral symptoms in PD mice but also significantly increase the expression of TH in SN and DA in the striatum. The neuroprotective effect of ECH (echinacoside) can potentially be correlated with anti-inflammation and anti-oxidation effects and promote the expression of endogenous nerve growth factors. Additionally, the present study found that the accumulation of α-synuclein (the core pathological product in PD (Parkinson's disease))had been significantly reduced in the EH group. Because it is relatively economical, effective, and safe, ECH (echinacoside) may provide a new therapeutic strategy for PD (Parkinson's disease) and even for other neurodegenerative illnesses including Alzheimer's disease.
echinacoside treat neurodegenerative illnesses including Alzheimer's disease
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