The main pathological changes inherent in Parkinson’s disease (PD) are degeneration and loss of dopamine neurons in the midbrain and formation of Lewy bodies. Many studies have shown that the pathogenesis of PD is closely related to endoplasmic reticulum (ER) oxidative stress. This study combined various traditional Chinese medicines to prepare Congrong Shujing granules (CSGs). The optimal dose combination of the ingredients was identified by experimental intervention in SH-SY5Y cells
Neurodegenerative disease has become the third most fatal disease globally after cardiovascular and cerebrovascular diseases and cancer, and Parkinson’s disease (PD) is the third most common neurodegenerative disease. In China, the prevalence of PD among those over 65 years of age is approximately 1.7% and is expected to affect approximately 5 million patients by 2030. Symptoms such as tremor, bradykinesia, rigidity, and ataxia gradually aggravate with the disease course, which lead to the loss of independent living and a financial burden for the family and society.
The main pathological features of PD are the progressive loss of dopamine neurons, apoptosis, and abnormal aggregation of proteins (Lewy bodies).
ERS refers to the disruption of ER morphology and functional homeostasis in the cells by internal and external factors resulting in an accumulation of unfolded or misfolded proteins in the ER. In response, cells initiate an unfolded protein response (UPR) to change the cellular transcription and translation levels. The burden of the cells is relieved by lowering the level of protein synthesis and increasing the expression of various molecular chaperones to assist protein folding [
Sustained and excessively strong ERS induces apoptosis by activating the three major downstream apoptotic signaling pathways [
Traditional Chinese medicine (TCM) has been widely used as an adjuvant therapy to PD treatment due to its relatively mild side effects and possible broader pharmacological effects. Congrong Shujing granule (CSG) is based on a combination of a TCM classic formula and clinical experience and has obtained a national patent (Patent number: 201410658358.3) in China. It originates from a classic TCM prescription, Dihuang Yinzi, recorded in the ancient Chinese medical book
This study aims to evaluate the effect of CSG on ERS and neuronal apoptosis in PD
In this study, two dose levels of each of the five herbs including
Drug-dose combination orthogonal design and efficiency calculation table L8 (27).
Experiment ID | Refined | Tree peony bark | Mean cell viability ( | |||
---|---|---|---|---|---|---|
A | 1 | 1 | 1 | 1 | 1 | 143.00 ± 11.79% |
B | 1 | 1 | 1 | 2 | 2 | 133.00 ± 6.25% |
C | 1 | 2 | 2 | 1 | 1 | 150.67 ± 5.13% |
D | 1 | 2 | 2 | 2 | 2 | 143.33 ± 6.03% |
E | 2 | 1 | 2 | 1 | 2 | 128.00 ± 7.55% |
F | 2 | 1 | 2 | 2 | 1 | 134.33 ± 13.65% |
G | 2 | 2 | 1 | 1 | 2 | 117.67 ± 8.74% |
H | 2 | 2 | 1 | 2 | 1 | 132.67 ± 2.89% |
Note: different levels of individual herbs:
Human neuroblastoma SH-SY5Y cells were purchased from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). Complete medium contained 445 ml DEME/F12, 50 ml fetal bovine serum, and 5 ml penicillin and streptomycin). Once the cells grew to 80–90% confluence, they were washed once with phosphate buffered saline (PBS) and incubated with 1 ml trypsin in the incubator after discarding the PBS. The complete medium was added to stop the reaction 2 min after the trypsinization, followed by pipetting the cells into a suspension and centrifuging at 1,500 rpm for 5 min. After centrifugation and supernatant removal, the cell pellet was resuspended in the complete medium and aliquoted into new culture flasks. The cells were divided into control, PD model, and drug intervention groups (compounds A–H). Cell viability was detected using the MTT assay after trypsinization and preparing a single-cell suspension in each group. By inoculating a density of 3,000 cells per well in a 96-well plate and preparing five replicates per group, MPP+ solution (1 mmol/ml) was added to each group after 24 h, followed by the addition of aqueous solution of different dose combinations to the cells for 24 h. After another 24 h, 20
A total of 70 specific-pathogen-free (SPF) male Sprague-Dawley (SD) rats, weighing 180 ± 20 g, were kept at the SPF-grade Experimental Animal Center of Fujian University of TCM, Fujian Province, China.
The rats were randomly divided into control, solvent, and PD model groups. Rats in the PD model group were intraperitoneal injected with rotenone and sunflower oil emulsion (1.5 mg/kg/day) for 14 days to establish a PD model, which was ranked according to the scoring criteria of the PD model. Rats with a score of ≥2 were selected as PD rats. Rats in the solvent group were subcutaneously injected with an equal volume of sunflower oil emulsion for 14 days. Then, the PD rats were randomly divided into a model group, low-dose group, middle-dose group, and high-dose group. The low-dose, the middle-dose, and the high-dose groups were treated by instillation with CSG once a day for 14 days. Meanwhile, the rats in the control, solvent, and model groups were given the same volume of normal saline for 14 days.
Experimental CSG daily dose in rat was calculated according to the daily dosage of 112 g in human and the human-rat body surface area ratio [
On the 14th day after modeling (before intervention) and the 14th day after CSG intervention, the suspension test was performed in the rats of each group. Rats were suspended on a horizontally straightened steel wire with a diameter of approximately 3 mm and a distance of 30 cm from the ground. A soft pad was placed on the ground to prevent injury from falling. During the test, the rats were not allowed to sit on the wire, and both forelimbs of the rats had to be suspended from the wire. In addition, the condition of front paws gripping on the wire and the duration of wire hanging of each rat was recorded and scored, with wire gripping continuously for 0–5 s scored as 0, 6–10 s as 1, 11–15 s as 2, 16–20 s as 3, 21–25 s as 4, 26–30 s as 5, and >30 s as 6. Scoring of each animal was recorded three times to take the mean value with an interval of approximately 2 min between each test.
The rats were anesthetized with urethane (1000 mg/kg bodyweight) by intraperitoneal injection, and no pain or discomfort was observed. This was because the anesthetic caused muscle relaxation and its other effects were mild. Rats were sacrificed by rapid cervical dislocation. Animals had little anxiety or pain by this way. We gave full consideration to the interests of animals; treated them humanely; prevented or reduced their stress, pain, and injury; respected animal life; stopped the barbarism against animals; and adopted the least painful method to deal with animals. The methods and purposes of animal experiments were in conformity with human ethical standards and international practices. Brain tissues of three rats from each group were collected for the detection of protein expression by rinsing off the fixative on the tissues and cryosectioning into brain slices.
In addition, three rats of each group were anesthetized intraperitoneally and perfused with 4% paraformaldehyde solution. After the perfusion was completed, the whole brain of each animal was carefully isolated and embedded in paraffin before sectioning. After washing, the precooled brain sections were washed three times in PBS, and the brain sections were blocked with goat serum for two hours. After discarding, the serum was discarded; anti-TH (1 : 400), anti-GRP78 (1 : 200), and anti-CDNF (1 : 200) primary antibodies were independently incubated with the brain sections at 4°C overnight. The remaining steps of the immunohistochemistry were performed in accordance with the manufacturer’s instructions of the Maxim immunohistochemistry kit (Maxim Biotechnologies, Fujian Province, China). The brain sections were then developed in 3,3′-diaminobenzidine (DAB) color development solution, counterstained nuclei were counterstained in hematoxylin dye, and the excessive dye was removed in 1% hydrochloric acid alcohol. After dehydrating in gradient ethanol solutions (3 min each) and clearing in xylene solutions I and II (3 min each), the brain sections were mounted with neutral gum. Once the mounted brain sections were dried up, they were subjected to imaging under a microscope. The relative integral optical density (IOD) values of TH, GRP78, and CDNF and the total area of the sections was measured: the average optical density value of the target protein = the IOD value of the target protein/total area of the tested brain region in the section.
For Western blot analysis, the substantia nigra in the midbrain of each group of rats was isolated and weighed, followed by complete homogenization and lysis for target protein detection. The anti-
Then, the brain tissue of rats was taken out, and the frozen sections were obtained. The frozen sections were put in citric acid buffer (pH 6.0) for antigen repair and then incubated with primary antibody (anti-TH mAb (Abcam, ab137869); anti-GRP78/Bip mAb (Abcam, ab21685)) and secondary antibody (goat anti-Rabbit IgG secondary antibody, Proteintech Group, 10285-1-AP), with PBS rinsing and DAPI redyeing. The slices were dried by antifluorescence quenching agent and put in Nikon fluorescence microscope for image acquisition.
We observed the number of neurons in the midbrain using DAPI staining, and we used the results to quantify apoptosis of neurons. The brain slices were stained in DAPI solution by covering the section and incubating at room temperature for 10 min. After removing the DAPI solution, the brain slices were washed in PBS three times (3 min each), followed by fluorescent microscopy at an excitation wavelength of 360 nm. We counted the number of neurons in the same area of view and compared the results from different groups.
The tissue samples were fixed with 2.5% glutaraldehyde, followed by rinsing three times in 1 M PBS and fixing with 1% osmic acid for 1 h. After rinsing in PBS and dehydrating with acetone, the tissue samples were embedded and sectioned into ultrathin slices, which were first stained with lead citrate for 10 min and washed with carbon dioxide-free double-distilled water three times and uranium acetate for 30 min. After washing in double-distilled water for three times and drying, the stained ultrathin slices were imaged by transmission electron microscope (TEM).
The experimental data were processed and analyzed by SPSS 24.0 (IBM SPSS Inc., Chicago, IL). The measurement data were presented as mean ± standard deviation. Comparison between multiple groups was performed by one-way analysis of variance (ANOVA). Analysis of the behavioral data was performed using a repeated measure ANOVA. Pairwise comparisons between groups were performed using Fisher’s least significant difference (LSD) method or Games–Howell method. The test level was
Table
Comparison of the therapeutic effects of CSG of different dose combinations on MPP+-induced
Chinese herbal medicine | Dose level | Number of experiments | Mean ± standard deviation | Confidence interval | |||
---|---|---|---|---|---|---|---|
1 | 3 | 142.50 ± 9.32 | 137.55 | 147.45 | 18.51 | <0.01 | |
2 | 3 | 128.17 ± 10.28 | 123.22 | 133.12 | |||
Refined | 1 | 3 | 134.58 ± 10.41 | 129.63 | 139.53 | 0.20 | 0.69 |
2 | 3 | 136.08 ± 13.96 | 131.13 | 141.03 | |||
1 | 3 | 131.58 ± 11.70 | 126.63 | 136.53 | 5.07 | 0.04 | |
2 | 3 | 139.08 ± 11.70 | 134.13 | 144.03 | |||
1 | 3 | 134.83 ± 15.30 | 129.88 | 139.78 | 0.09 | 0.77 | |
2 | 3 | 135.83 ± 8.37 | 130.88 | 140.78 | |||
Tree peony bark | 1 | 3 | 140.17 ± 11.06 | 135.22 | 145.12 | 8.42 | 0.01 |
2 | 3 | 130.50 ± 11.45 | 125.55 | 145.45 |
Model
Morphology and OD value of the cells in the drug efficacy screening experiment. (a–j) The effects of different TCM compounds on SH-SY5Y cell survival. (k) The statistical data of OD values of the groups. Compared with the PD model, “∗” indicates
As shown in Tables
Repeated measures analysis of variance of suspension experiment (mean ± standard deviation).
Group | Before treatment | 5 d | 10 d | 14 d | Comparison of different timepoints in the same group | ||
---|---|---|---|---|---|---|---|
Control | 9 | 2.30 ± 0.51 | 2.22 ± 0.46 | 1.92 ± 0.32 | 1.70 ± 0.13 | 15.55 | <0.01 |
Solvent | 9 | 2.37 ± 0.48 | 2.23 ± 0.49 | 1.91 ± 0.36 | 1.57 ± 0.21 | 24.34 | <0.01 |
Model | 9 | 0.37 ± 0.11 | 0.39 ± 0.10 | 0.43 ± 0.17 | 0.47 ± 0.12 | 2.62 | 0.116 |
Low dose | 9 | 0.34 ± 0.08 | 0.41 ± 0.85 | 0.61 ± 0.16 | 0.69 ± 0.15 | 55.87 | <0.01 |
Middle dose | 9 | 0.44 ± 0.12 | 0.51 ± 0.15 | 0.84 ± 0.05 | 0.87 ± 0.11 | 44.04 | <0.01 |
High dose | 9 | 0.38 ± 0.08 | 0.48 ± 0.09 | 0.81 ± 0.05 | 0.88 ± 0.07 | 95.63 | <0.01 |
Comparison of groups at the same timepoint | F | 101.41 | 89.78 | 80.39 | 113.39 | - | - |
<0.01 | <0.01 | <0.01 | <0.01 | - | - |
Intra- and intergroup variance of suspension experiments.
Source of variation | SS | df | MS | |||
---|---|---|---|---|---|---|
Intragroup variation | Time | 0.11 | 1.41 | 0.08 | 1.90 | 0.17 |
Time | 9.04 | 7.03 | 1.29 | 32.01 | <0.01 | |
Error | 2.71 | 67.45 | 0.04 | |||
Intergroup variation | Group | 105.21 | 5.00 | 21.04 | 112.61 | <0.01 |
Error | 8.97 | 48.00 | 0.19 |
SS: sum of square; df: degree of freedom; MS: mean square.
Ethology and immunostaining of rats. (a) Score of the suspension experiment after different days of treatment. (b
Immunohistochemistry showed that CSG interventions increased the TH, GRP78, and CDNF expressions (Figures
Protein expression in different groups as indicated by Western blot analysis. (a
TEM and DAPI staining showed fewer apoptotic nerve cells in the middle-dose group than in the model group (Figures
Numbers of neurons and morphological observations of the endoplasmic reticulum. (d
Rotenone is a widely used insecticide that selectively inhibits the activity of mitochondrial respiratory chain complex I in neurons, causing degeneration and apoptosis of dopaminergic neurons. It was first used by Heikkila et al. [
Some research results indicate that ERS and UPR are widely involved in the pathophysiology of PD. PERK, IRE-1, and ATF6 are important molecules in the three signaling pathways of ERS. In the early stages of ERS, UPR is a protective program. However, after chronic activation, it triggers apoptosis. Under the influence of ERS, PERK separates from its molecular chaperone, GRP78, and activates it, causing downstream eIF2 phosphorylation and inactivation, which stops most protein synthesis in the cell [
In this study, GRP78 expression was downregulated in the rotenone-induced PD model. After 14 days of CSG interventions, the expression of GRP78 in the substantia nigra of the midbrains of rats in middle- and high-dose intervention groups was significantly upregulated. Expression of the key functional enzyme, TH, and CDNF in dopaminergic neurons was increased, while the JNK and phosphorylated c-JUN expression in the substantia nigra of the midbrains of rats and the apoptosis were significantly reduced. TEM study on the ultrastructure of ER showed that CSG improved ER fragmentation and degranulation in neurons of the PD rat model. In addition, the numbers of ER and mitochondria in the area of substantia nigra of the PD rat model were increased after CSG intervention, suggesting that the intracellular life activity of neurons affected by neurotoxin was recovered. Given the close relationship between GRP78-downregulated ERS and the CDNF and JNK pathways, this study demonstrated that CSG may reduce ERS by increasing GRP78 expression and reducing ERS level in the substantia nigra, thereby triggering the protective effects on the PD rat model.
All data in this manuscript are available from the corresponding author for reasonable reasons and research needs.
All procedures involving animals were conducted in accordance with the ethical standards of the institution, comparable ethical standards, and the 1964 Helsinki declaration and its later amendments.
The authors declare that there are no conflicts of interest.
Qian Xu, Shasha Yang, Fangzhen Wu, Jianan Zhong, Lanfang Tang, and Yao Lin are responsible for the experiments and data curation. Xuefeng Hu and Jing Cai contributed to project administration, review, and editing of the paper.
The work was supported by the Creation of Major New Drugs for Major National Science and Technology Projects (no. 2019ZX09301154), National Natural Science Foundation of China (nos. 81904263 and 81804156), Fujian Traditional Chinese Medicine Research Project (nos. 2017FJZYLC202 and 2017FJZYLC501), and 2018 Outstanding Young Scientific Research Project in Fujian Province.