Tenuifolin suppresses mitophagy via the PINK1/Parkin pathway as a potential therapeutic approach for Parkinson's disease models

doi:10.21203/rs.3.rs-3808567/v1

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Research Article

Tenuifolin suppresses mitophagy via the PINK1/Parkin pathway as a potential therapeutic approach for Parkinson's disease models

https://doi.org/10.21203/rs.3.rs-3808567/v1

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Objective

The occurrence of Parkinson's disease (PD) may be caused by mitophagy, and we tried to find out the clues that Tenuifolin (TEN) alters mitophagy.

Methods

The effects of TEN on mitophagy and PINK1/Parkin pathway were detected by ELISA, RT-qPCR, western blot and electron microscopy in rotenone-induced PD rats and PC12 cells.

Results

ROT can establish a rat model of PD, with PD-related behavioral and brain pathological changes. TEN administration can improve the appearance and behavior of PD model rats, reduce the expression of Parkin, PINK1, Beclin-1, LC3II/I and other related autophagy proteins in the brain tissue of PD model rats and PC12 cells, alleviate the damage of cell mitochondria, and protect the brain nerve.

Conclusion

PINK1/Parkin pathway may be one of the mechanisms of TEN to treat PD via alleviating the damage of cell mitochondria and protecting the brain nerve, and it provides a new way to treat Parkinson's disease.

TEN

PINK1

Parkin

Mitophagy

Parkinson's disease is a common neurodegenerative disease in elderly patients, which is extremely destructive and irreversible[1, 2]. Its incidence ranges from 41 per 100,000 at the age of 40 to more than 1900 per 100,000 over the age of 80[3]. The main symptoms of Parkinson's disease are involuntary tremors, gait stiffness, and decreased motor ability[4]. At present, the treatment of Parkinson's is still based on levodopa preparations, which are commonly used clinically, but it will produce obvious side effects, which are proportional to the dose. The higher the dose, the more serious the side effects[5, 6]. Traditional Chinese medicine (TCM) has been used for the treatment of dyskinesia corresponding to the clinical manifestations of PD for more than two thousand years[7–10]. In recent years, pharmacological studies of traditional Chinese medicine have shown that traditional Chinese medicine has shown potential clinical efficacy in alleviating PD symptoms[11–14].

The pathogenesis of Parkinson's disease is still unclear, among which mitochondrial autophagy is considered to be the most important pathogenesis of PD[15–17], and has become a research hotspot in recent years[18]. Autophagy is a degradation process that exists in cells. It can effectively recycle damaged organelles and misfolded proteins in cells. This process maintains a high degree of stability in cells[19]. There are many ways to activate autophagy, but the most classic way is to activate autophagosomes and lysosomal channels. In addition, autophagy can also regulate mitochondria by regulating the state of cells[18, 20]. The most common type of organelle autophagy is mitophagy, through which damaged, aging, and necrotic cells can be identified for removal, destruction, and phagocytosis. This selective removal method is mitophagy (syndrome)[21, 22].Parkin gene has an inhibitory effect on mitophagy, thereby reducing mitochondrial lysis[23, 24].PINK1 gene will affect the initiation and progress of mitophagy regulated by PINK1/Parkin, such as when the kidney is in a state of less blood, monocytes and some neutrophils quickly recognize damaged cells, and lysosomal enzymes are quickly released through the cell membrane while penetrating into the blood, which in turn leads to renal tubular epithelial cell damage[25, 26].

According to "Shen Nong's Materia Medica", the traditional Chinese medicine Polygala is bitter in taste, pungent in nature, warm in nature, and has the heart, kidney, and lung meridians[27]. The plant Polygala belongs to Polygala tenuifolia Willd or the dried root of Polygala sibirica L, which has the functions of calming the nerves, communicating with the heart and kidney, resolving phlegm and relieving cough, and can effectively improve the condition of patients[28]. Recent studies have shown that polygala can not only be used to prevent and treat poor sleep caused by heart-kidney disharmony, but also has anti-dementia, anti-depression, and maintenance of cardiovascular and cerebrovascular functions[29, 30]. In addition, Polygala saponin is also an important component of Polygala sedative and intellectual function, it can anti-aging, control cell apoptosis, promote consciousness and protect brain mitochondrial damage[31]. After alkali hydrolysis and attenuation, the properties of Polygala saponins are more stable than the total saponins of Polygala, and the toxic and side effects are greatly reduced[32]. In addition, research by You Fuling and others found that TEN can significantly increase the activity of mitochondrial SOD and GSH-Px, significantly reduce the concentration of MDA in hippocampal nerve cells, eliminate excessive free radicals in the human body, and effectively protect the basic shape of mitochondria, thereby improving the body's immunity and strengthening the immune system function of organisms.

In summary, this study intends to use the Parkinson's injury model established by ROT, and to explore the role of TEN in the development of PD and its role. The mechanism, from the perspective of mitophagy, explains that mitophagy mediated by the PINK1/Parkin pathway improves ROT-induced Parkinson's disease rats, thereby inhibiting the progression of PD disease.

Grouping and modeling of animals

Forty SPF male Sprague-Dawley (SD) rats, weighing 200-220 g, were randomly divided into five groups: control group, model group, and TEN low, medium, and high dose groups. The PD model was established by subcutaneous injection of ROT on the neck and back for 14 consecutive days. The behavioral changes of the rats were observed, including general behavioral tests, balance beam tests and roller tests. TEN (2, 4, and 6 mg/kg) was intraperitoneally injected for 14 days after successful modeling. Experimental animals were provided by the Animal Experiment Center of Southwest Medical University (License: SYXK(Sichuan)2018-065). The experimental plan was approved by the Animal Ethics Committee of Southwest Medical University (batch number: NO.202111126-046).

Balance beam test

The apparatus was a cylindrical wooden stick (50 cm in length and 1.0 cm in diameter), which was fixed horizontally at a height of 50 cm on the ground. The starting and ending points of the stick were marked, and the rats were allowed to crawl on the crossbar.

Roller test

The roller was maintained at a constant speed of 3 RPM before testing. The roller was accelerated to 40 RPM for no more than 300 seconds, and the time the rat remained on the roller was recorded (the experiment was terminated when a foot was dropped) three times a day (9 a.m, 3 p.m, and 6 p.m.). The patients who stayed on the roller for 150 s (including 150 s) in each group were included in the study.

HE staining of brain and liver tissues

Rat brain tissue and liver were fixed in 4% paraformaldehyde solution, dehydrated, transparent, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE).

Cell Culture

PC12 cell was obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA) Rat adrenal pheochromocytoma (poorly differentiated). PC12 was cultured with medium supplemented with 10% fetal bovine serum, 50 U/mL penicillin and 50 μg/mL streptomycin (Invitrogen, Scotland, UK) in a 5% humidified CO2 incubator at 37 °C. PC12 cells were divided into control group, ROT group, ROT+TEN group, ROT+TEN+3-MA group, and ROT+TEN+positive drug group.

Cell viability Assays

Cell viability measurement was performed using the CCK-8 assay (Biyuntian, BS350B) according to the instruction manual. The percentage of cell viability was calculated using the following formula: Cell viability (%) = (measured value - blank value)/(control value - blank value) × 100%.

ELISA

The GSH-Px, SOD and MDA kits used were purchased from Nanjing Jiancheng Biotechnology Research Institute, and the corresponding content of cell activity was detected at the absorbance wavelengths of 412 nm, 550 nm, and 532 nm according to the instructions.

JC-1

Flow cytometry test kit purchased from Shanghai Biyuntian Biotechnology Co, Ltd.Culturing cells using the same method described above. Dyeing machine according to the kit[33].

TEM

Preparation of samples for electron microscopic examination, 3% glutaraldehyde fixation, acetone dehydration, infiltration and embedding of specimens, ultrathin sectioning, staining and observation[34, 35].

Western Blot

After drug treatments, cells were harvested and lysed in RIPA buffer from Cell Signaling Technologies Inc. (Beverly, MA, USA). Protein concentrations were measured by Bradford reagent (Bio-Rad, Hercules, CA, USA). The cell lysates were then resolved by SDS-PAGE. After electrophoresis, the proteins from SDS-PAGE were transferred to nitrocellulose membrane which was then blocked with 5% non-fat dried milk for 60 min. The membrane was then incubated with corresponding primary antibodies overnight at 4 °C. After that, the membrane was further incubated with HRP-conjugated secondary antibodies for 60 min[36]. Finally, protein bands were visualized by using the ECL Western Blotting Detection Reagents (Invitrogen, Paisley, Scotland, UK). Band intensities were quantified by using the software ImageJ (ImageJ 1.46r; National Institutes of Health, Bethesda, MD, USA).

Real Time PCR Analysis

In brief, PC-12 cells transfected with EGFP-HDQ 74 were incubated with TEN for 16 h before RNA extraction[37, 38]. Total RNA was extracted from PC-12 cells by using FavorPrep™ Total RNA purification mini kit (Favorgen, Ping Tung, Taiwan). cDNA were synthesized by performing reverse transcription using SuperScript® VILO™ Master Mix (Invitrogen, Grand Island, NY, USA). Real-time PCR were carried out on ViiA™ 7 Real Time PCR System (Applied Biosytems, Grand Island, NY, USA) using the FS Universal SYBR Green Master Rox (Roche, Indianapolis, IN, USA) according to manufacturer’s instructions. PCR was done by using primers 5'-ATG AAG GCC TTC GAG TCC CTC AAG TCC TTC-3' and 5'-GGC GGC TGA GGA AGC TGA GAA-3' as described[39, 40].

Statistical Analysis

The results were expressed as means±SD. as indicated. The difference was considered statistically significant when the p-value was less than 0.05. Student’s t-test or one-way ANOVA analysis was used for comparison among different groups.

ROT modeling induced PD-related behavioral changes in SD rats

After a week of drug ROT modeling treatment, the hair color of the ROT group changed, with decreased activity, decreased autonomous movement, and decreased anti capture reflex. The most severe and prominent cases occurred between 7-14 days. Considering the high mortality rate of rats when the dose was 2 mg/kg, 1.5 mg/kg was chosen as the subsequent modeling dose.

PD model rats were prolonged through the balance beam

The balance beam test results showed that (Figure 1A and Figure 1B) rats in the control group could successfully pass the balance beam within 10 sec, with a score of (6.00 ± 0.03) points, while the average 24-hour score of the model group was (3.85 ± 0.60) points. There was a significant difference in the time to pass the balance beam before and after modeling, with statistical significance (P<0.01).

PD model rats on the roller residence time was shortened

The roller test was further used to detect whether the motor coordination of the rats had changed before and after modeling . The experiment found that ( Figure 1C) the average residence time on the roller of rats in the normal group was (26.20 ± 2.251) s, while the average residence time on the roller of rats in the model group (1.5 mg/kg and 2 mg/kg) was (17.39 ± 1.669) s and (16.39 ± 2.643) s, respectively. The time was shorter than that of the control group, with significant differences compared to the normal group (P<0.01), indicating that after ROT modeling, the motor coordination of SD rats was decreased.

TEN improved the pathological changes of brain tissue in PD rats and did not damage the liver

In this study, the size change of brain tissue before and after modeling is not significant (Figure 2C).After TEN drug intervention, the pathological changes of the brain can be improved (Figure 2A). Orphology of neurons in the substantia nigra of brain tissue was improved, with enlarged nuclei, distinct nucleoli, more consistent staining, clearer glial cell structures, no evidence of neurophagocytic cells, almost no hyperemia of stroma vessels, widening of peripheral space, and appearance of lymphocyte surrounding tubes. These improvements were superior to those in the ROT group, with the high-dose group (6 mg/kg) having the most significant improvement in brain cells. Subsequently, the hepatotoxicity of TEN on experimental animals was tested to comprehensively evaluate the drug value. The results showed that , in the ROT group, there were more punctate necrosis and nuclear pyknosis of hepatocytes. After intervention with different concentrations of TEN, the cells gradually returned to a radial arrangement, without significant hepatotoxicity. In the high-dose group (6 mg/kg), the hepatic cords were arranged neatly, and the morphology of hepatocytes was basically restored.

TEN reduced the expression levels of Parkin and PINK1 mRNA

In vitro experiments confirmed that TEN can relieve mitochondrial autophagy and improve mitochondrial function, thereby slowing down the occurrence of PD. Next, the effect of TEN on the occurrence of related mitophagy in brain tissue was further detected. Then, the changes of the mRNA levels of Parkin and PINK1 in the brain tissue of different experimental groups were detected in the animal experiments, and it was found that (Figure 4D and Figure 4E) in the ROT group, the mRNA expressions of Parkin and PINK1 were increased compared with the control group, and the difference was statistically significant (P <0.001).

Similarly TEN decreased the expression of Parkin and PINK1 mRNA in PC12 cells. The results showed (Figure 3F and Figure 3G) that compared with the control group, the ROT model group could increase the expression levels of PINK1 and Parkin mRNA; compared with the ROT model group, the TEN group had a significant down-regulation effect on the expression levels of PINK1 and Parkin mRNA induced by ROT. The results showed that TEN could reduce the expression levels of PINK1 and Parkin mRNA induced by ROT with statistical significance.

TEN drug concentration screening and its effect on PC12 cell viability

As shown in Figure 3A, compared with the control group, the activity of TEN above 50 μM decreased in vitro (P<0.01), and all of them could significantly inhibit the proliferation of PC12 cells, while TEN 25 μM had no significant effect on PC12 cells. According to the results of this experiment, the settings of 6.25 μM, 12.5 μM, and 25 μM were selected for subsequent experiments.

TEN attenuated mitochondrial damage caused by ROT

PC12 cells were treated with different concentrations of TEN (6.25 μM, 12.5 μM and 25 μM) for 24 h. The ultrastructural changes of PC12 cells were observed by transmission electron microscope. The results showed that (Figure 2B), the chromatin in the nucleus was evenly distributed, and the organelle related structures could be clearly observed in the cytoplasm in the control group. In the TEN6.25 μM group, most of the mitochondria in the nucleus were swollen in the cytoplasm, and a small amount of autophagy and lipid droplets were observed in the cells. In the TEN12.5 μM group, there was no obvious abnormality in the nucleus, only a few mitochondria in the cytoplasm were slightly swollen, and a small amount of lipid droplets were occasionally seen. There were no obvious abnormalities in the nucleus of the TEN25 μM group, and most of the mitochondria were slightly swollen in the cytoplasm, and a small amount of autophagy and lipid droplets were also observed.

TEN alleviated ROS generation in PC12 cells with ROT-induced

The experimental results showed in Figure 3B,C and D,TEN could increase the activity of GSH-Px and SOD in PC12 cells, and reduce the MDA content. Compared with the control group, the activities of GSH-Px and SOD could be inhibited after ROT treatment. After intervention with different concentrations of TEN, there was no significant difference in the results between the low concentration group TEN (6.25 μM) and the ROT group. Both the medium concentration group TEN (12.5 μM) and the high concentration group TEN (25 μM) could significantly inhibit the ROT effect and restore the activity of GSH-Px and SOD in PC12 cells, and the medium concentration group TEN (12.5 μM) had the best effect (P <0.01). Meanwhile, the detection of PC12 culture supernatant revealed that the content of MDA in culture supernatant increased significantly after ROT treatment, indicating that ROT could promote lipid peroxidation in PC12 cell membrane (P< 0.01).

TEN increased the mitochondrial membrane potential in PC12 cells

At the same time, mitochondrial membrane potential (MMP) was used to detect the effect of TEN on mitochondrial function. The results showed that (Figure 3E) the percentage of JC-1 in the right lower quadrant of the PC12 cells in the ROT group was higher than that in the control group, indicating that JC-1 existed as a monomer in the cells, giving off green fluorescence, and the mitochondrial membrane potential decreased, suggesting that ROT caused damage to the mitochondria of PC12 cells (P<0.001). After TEN treatment, the percentage of the right lower quadrant decreased, indicating that mitochondrial membrane potential increased and mitochondrial function gradually recovered. At the same time, the mitochondrial membrane potential was significantly increased by TEN at different concentrations (P<0.01).

TEN downregulated the expression of autophagy proteins in the PINK1/Parkin pathway

The results showed that (Figure 4A) ROT treatment increased the protein expression of Becline-1 in the brain cells of SD rats (P<0.01). Different concentrations of TEN could down-regulate the expression of Becline-1, of which TEN (4 mg/kg) had the most significant effect. Compared with the control group(Figure 4B), the protein expressions of PINK1 and Parkin in the brain of the model group were up-regulated (P<0.01). TEN treatment down-regulated the expression of Parkin and PINK1, and consistent with Becline-1, the medium dose of TEN (4 mg/kg) group had the most significant effect (P<0.01).

The expression of autophagy markers p62 and LC3II/I were further examined in the brain tissues of PD rats. The results showed that (Figure 4C) compared with the control group, the expression of LC3II/I protein in the ROT group was increased, while compared with model group, the expression of LC3II/I was decreased and the expression of p62 was promoted after TEN treatment.

In vitro experiments showed that (Figure 5A) the expression of Becline-1 was up-regulated after ROT treatment in PC12 cells.And the expression of Becline-1 was down-regulated, at the same time TEN had a synergistic effect with autophagy inhibitors 3-MA and RAPA.

PINK1 and Parkin, two autophagy-related proteins in the autophagy signaling pathway that regulate PD, were further detected (Figure 5B). In PC12 cells treated with different drugs for 24 h, ROT could up-regulate the expression of PINK1 and Parkin. TEN can down-regulate the expression of PINK1 and Parkin, and TEN has a synergistic effect with 3-MA and RAPA.

Meanwhile, LC3II/I and P62, two important proteins in the autophagy signaling pathway, were also detected. The results showed that (Figure 5C) the protein expression of LC3II/I in rat PC12 cells in the ROT group was higher than that in the control group. After TEN treatment, the expression of LC3II/I was significantly down-regulated.

Traditional Chinese medicine containing TEN has been widely used in the treatment of PD in China. TEN is the main component of Polygala, but the role of TEN in Parkinson's disease is still unclear. Therefore, it is of great clinical significance to further study the role and mechanism of TEN. It has been shown that TEN can inhibit nerve injury through SHP-2-mediated mitophagy. Therefore, we explored the effect of TEN on ROT-induced Parkinson's disease and the underlying mechanisms.

Our results showed that ROT induced mitochondrial damage in brain tissue and neurons, which led to the increase of intracellular autophagosomes, while TEN treatment clearly alleviated the above damage. Meanwhile, PINK1/Parkin pathway activation was found to have a significant protective effect on brain function. Parkin transferred from the cytoplasm to the mitochondria to activate p62, and p62 co-activated with the autophagy protein LC3 to activate the mitophagy pathway[41, 42]. When the double-layer autophagic membrane wrapped the damaged organelles, autophagic degradation began[25, 43, 44]. Therefore, we hypothesize that TEN alleviates mitochondrial damage through PINK1/Parkin pathway to alleviate PD progression. We found that TEN downregulated the expression of autophagy proteins in PINK1/Parkin pathway, increased mitochondrial membrane potential and attenuated mitochondrial damage. Mitochondria are the most important energy providers in the body. When mitochondria are damaged, energy supply is dysregulated, which can lead to oxidative stress damage[45, 46]. We found that the oxidative stress injury in PC12 cells was alleviated after TEN intervention. These beneficial effects of TEN may be a potential drug for the treatment of Parkinson's disease.

Although our study on the animal and cell level confirmed TEN protective effect of Parkinson's disease, but we still need more clinical trials to support our point of view. In addition, our study did not explore how mitochondria contribute to the development of PD and the mechanism by which TEN inhibits the expression of autophagy proteins, which is a direction worthy of our exploration.

This study indicated that TEN attenuated ROT-induced neuronal cell damage both in vivo and in vitro. The protective mechanism may be to inhibit mitochondrial autophagy by suppressing autophagy protein expression through the PINK1/Parkin pathway.TEN can be considered as a new potential drug for the treatment of Parkinson's disease.

Acknowledgments

We would like to express our gratitude for the experimental facility provided by the Luzhou Key Laboratory for the Prevention and Treatment of Cardiovascular and Cerebrovascular Diseases with Integrated Traditional Chinese and Western Medicine.

Authors’ contributions

XZ, MY and HC carried out the data analysis and wrote the manuscript. TW and RZ performed the animal studies. GH and DW were responsible for quality assurance of the studies. HL and HS conducted pathological analysis. SY, YZ and QS guided the project.

Funding Information

The work was supported by the Sichuan Provincial Science and Technology Departm (No. 2021YFH0150), the Sichuan Provincial Administration of Traditional Chinese Medicine (No. 2021MS107), the Luzhou Science and Technology Bureau (No. 2021LZXNYD-Z09) and the Sichuan Provincial Science and Technology Departm (No. 2022YFS0635).

Data Availability

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Ethics approval and consent to participate

The animal study was reviewed and approved by the Animal Experiment Center of Southwest Medical University (License: SYXK(Sichuan)2018-065), China. All the animal experiments were conducted in accordance with the guidelines of the institutional bioethical committee. The study was reported in accordance to ARRIVE guidelines (https://arriveguidelines.org).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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Tenuifolin suppresses mitophagy via the PINK1/Parkin pathway as a potential therapeutic approach for Parkinson's disease models

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Abstract

Objective

Methods

Results

Conclusion

Figures

Bacground

Methods

Grouping and modeling of animals

Balance beam test

Roller test

HE staining of brain and liver tissues

Cell Culture

Cell viability Assays

ELISA

JC-1

TEM

Western Blot

Real Time PCR Analysis

Statistical Analysis

Results

ROT modeling induced PD-related behavioral changes in SD rats

PD model rats were prolonged through the balance beam

PD model rats on the roller residence time was shortened

TEN improved the pathological changes of brain tissue in PD rats and did not damage the liver

TEN reduced the expression levels of Parkin and PINK1 mRNA

TEN drug concentration screening and its effect on PC12 cell viability

TEN attenuated mitochondrial damage caused by ROT

TEN alleviated ROS generation in PC12 cells with ROT-induced

TEN increased the mitochondrial membrane potential in PC12 cells

TEN downregulated the expression of autophagy proteins in the PINK1/Parkin pathway

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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