How does echinacoside from cistanche benefits patients with Parkinson’s disease
Mar 06, 2022
Contact: emily.li@wecistanche.com
Part Ⅲ：Mechanism Of Autophagy Regulation in MPTP-Induced PD Mice Via The MTOR Signaling Pathway By Echinacoside
The occurrence of Parkinson’s disease (PD)is related to many factors, but the main pathological mechanisms seem to focus on oxidative stress, mitochondrial dysfunction, and abnormal protein aggregation. All these mechanisms are highly correlated with autophagy.16 For living organisms, autophagy plays an important role. It plays a key role in removing the misfolded proteins, the abnormally aggregated proteins, and damaged organelles. It has been reported that the up-regulation of autophagy can eliminate some disease-related proteins in PD (Parkinson’s disease), such as α-synuclein, ubiquitin, and other misfolded proteins, thereby reducing the incidence of PD (Parkinson’s disease). Meanwhile, studies have also pointed out that the down-regulation of autophagy can lead to mitochondrial dysfunction, mitochondrial rupture, deposition of insoluble SNCA/α–synuclein, and ubiquitin proteins.10 Therefore, it is important to regulate autophagy to maintain normal function.
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Preliminary studies of our research group also confirmed in both Vivo and Vitro studies that Echinacoside had a certain neuroprotective effect9,20–23 and may reduce the expression of α-synuclein. To further explore the specific mechanism of Echinacoside in the clearance of α-synuclein, we conducted a more in-depth study on the neuroprotective mechanism of ECH in MPTP-induced PD (Parkinson’s disease) mice from the perspective of autophagy. In the present study, the neurobehaviors in mice of each group were evaluated first. It was found that compared with the M group, the climbing time of mice in the EH group was significantly shorter, and the latency to fall and the stride length of the fore and hind limbs were significantly extended. The ability of autonomous activity in mice in the EH group was also significantly increased. The results of HPLC showed that the content of monoamine neurotransmitters increased significantly in the EH group compared with the M group, especially the expression of DA and DOPAC. The results of Western blot also revealed that the expression of TH in the substantia nigra increased noticeably in the EH group compared with the M group. While the above neurobehavioral functions deteriorated significantly in the CQ group and WO group, the content of DA and TH were also significantly lower than that in the EH group, and the mortality was significantly higher. According to the results of the present study, the mortality in the autophagy inhibitor groups was higher than in the other groups. The mortality in the CQ group was 28%, and that in the WO group was 12%, while there were no deaths in the other groups. To verify the effect of autophagy inhibitors on Echinacoside, double immunofluorescence of TH and α-synuclein staining, together with the protein immunoblotting of α-synuclein, were adopted to observe the expression of α-synuclein. The results confirmed that Echinacoside did significantly reduce the expression of α-synuclein compared with the M group. However, with the application of autophagy inhibitors, the expression of α-synuclein increased sharply. Studies have pointed out that the toxic effects of MPTP are closely related to the appearance of α-synuclein because the mice of the α-synuclein knockout show a high degree of tolerance to the toxicity of MPTP.24 This might indirectly suggest that for the MPTP-induced PD (Parkinson’s disease) mice models, the higher the expression of α-synuclein, the stronger the toxic effect of MPTP on mice. In the present study, the mortality of mice in the autophagy inhibitor group was significantly higher than that in other groups, which might be correlated with the increased expression of α-synuclein and the decreased tolerance to the toxic effects of MPTP. Therefore, we inferred that the neuroprotective effect of Echinacoside might be correlated with the promotion of autophagy, and autophagy inhibitors might significantly increase the expression of a-synuclein, which might be correlated with the death of mice.
As a key marker of autophagy, the light chain 3 of the microtubule-associated protein 1 (LC3) is widely used to detect the occurrence of autophagy by immunofluorescence or Western blot.P62, also known as SQSTM1, is involved in the pathogenesis of a variety of neurodegenerative diseases. It can link the ubiquitin proteins to LC3 and degrade through autophagy. The protein expression level of P62 can reflect the completion of autophagy flow.2 To further investigate the regulation mechanism of Echinacoside on MPTP-induced autophagy in PD (Parkinson’s disease) mice, the expression of autophagy-related indicators such as Beclin 1, LC3 and P62 were measured in mice in each group. It was found that the expressions of Beclinl and LC3-Ⅱ increased significantly in the M group compared with the N group, together with an obvious increase in the number of autophagosomes. These suggested that the level of autophagy in mice in the M group was increased. However, the level of P62 did not decrease with the increase of autophagy. Instead, it increased significantly, indicating that the entire autophagy process was not completed normally. That meant there existed an obvious autophagic flow disorder in the MPTP group, which was consistent with reports in the literature.26,27 Rapamycin can increase the synthesis of lysosomes and promote the clearance of autophagosomes. In the present study, the classic autophagy inducer rapamycin was adopted as a positive control, and the classic autophagy inhibitors chloroquine and wortmannin were used as controls. Immunofluorescence and Western blot were used to observe the expression level of LC3. The expression of Beclin l was measured by Western blot, and the expression of autophagosomes in each group of mice was observed by a transmission electron microscope. The results revealed that compared with the M group, the expressions of Beclinl and LC3-II increased in the Echinacoside group and RA group, while the levels of P62 and α-synuclein decreased significantly, indicating that in these two groups, the autophagic flow disorder improved significantly than that in MPTP-induced PD (Parkinson’s disease) mice. The expressions of Beclin 1, LC3-II, and the number of autophagosomes in the WO group decreased, accompanied by the increased levels of P62 and a-synuclein. This correlated with the failure of clearance of the autophagy substrate and pathological products over time due to autophagy inhibition. The expression of LC3-II and the number of autophagosomes in the CQ group increased significantly, along with an increase in the levels of P62 and a-synuclein, which was correlated with the disorder of autophagy flow.
It is known that mTOR kinase is a key regulatory site for autophagy, and it exists in the cell as two complexes with different functions, mTORCl, and mTORC2. mTORCl, which is sensitive to rapamycin, is the main negative feedback regulator of autophagy, while mTORC2, which is not sensitive to rapamycin, can regulate the activation of protein kinase AKT. The activated AKT can act on the mTORCl complex; therefore, AKT may act as the connection point between mTORC1 and mTORC2.28 Through the present study, it was found that, like rapamycin, Echinacoside could up-regulate autophagy by inhibiting the expression of mTOR and also up-regulate the expression of p-AKT/AKT. We speculated that Echinacoside might inhibit the expression of mTORC1, thereby up-regulating autophagy, achieving the effect of eliminating a-synuclein and P62, and then activating AKT through mTORC2 to promote the survival of dopamine neurons. However, chloroquine is not the inhibitor of mTOR-dependent induction of autophagy and dephosphorylation of the regulator, so it will be avoided in future studies as experimental means for mTOR-dependent inhibition of autophagy.
echinacoside treat PD (Parkinson’s disease)
Summary
In short, targeted at mTOR, the development of drugs that can both increase clinical efficacy and reduce clinical toxicity may be the direction of future research. Echinacoside has many advantages, such as safety, non-toxicity, low side effects, and low price. Our preliminary animal experiments had shown that Echinacoside could inhibit the mTOR pathway, increase autophagy, and promote the decrease of P62 and the elimination of pathological products such as α-synuclein. This could bring new treatment strategies for traditional Chinese medicine to intervene in PD (Parkinson’s disease). However, considering the insufficient sample size and no relevant cell experiments for verification, this would be the direction of our further research.
Ethics Approval and Consent to Participate: All experiments were evaluated and approved by the Ethics Committee of Nanjing Hospital of Chinese Medicine and complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals(KY2017102).
Acknowledgments: We are particularly grateful to all the people who have given us help with our article.
Funding: This study was funded by:1. Nanjing Health Science and Technology Development Special Fund Grant (YKK18134);2. Jiangsu Provincial Bureau of Traditional Chinese Medicine "Chinese Medicine Brain Diseases" Key Discipline Cultivation Project (NBPY201704)3. The Nanjing University of Chinese Medicine "Jiangsu University Nursing Advantageous Discipline Construction Project Funding Project"(2019YSHL098);4. Nanjing "Thirteenth Five-Year Plan"Famous Chinese Medicine Doctor (Kong-Jiang Liu) Studio Construction Project (LKJ-2017-NJ).5. Nanjing"Thirteenth Five-Year Plan"Famous Chinese Medicine Doctor (Jing-Qing Wang)Studio Construction Project (WJQ-2019-NJ). The funding body had no role in the design of the study and collection, analysis, and interpretation of data, and in writing the manuscript.
Disclosure: The authors declare that they have no competing interests.
References
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