Abstract
As a classical prescription of Traditional Chinese medicine, the Jia-Jian-Di-Huang-Yin-Zi (JJDHYZ) decoction has long been used to treat movement disorders. The present study evaluated the effects of JJDHYZ on dopaminergic (DA) neurons and their survival-enhancing microenvironment as well as the possible mechanisms involved using a mouse model of Parkinson’s disease. In MPTP-lesioned mice, a high dosage of JJDHYZ (34 g/kg/day) attenuated the loss of DA neurons, reversed the dopamine depletion, and improved the expression of glial-derived neurotrophic factor (GDNF) compared to the untreated model group. JJDHYZ also protected the ultrastructure of the blood-brain barrier (BBB) and tight junction proteins by inhibiting the activation of microglia and astrocytes besides the increase in three types of matrix metalloproteinases in the substantia nigra. In conclusion, the JJDHYZ-high dosage (JJDHYZ-H) group exhibited the neuroprotection of DA neurons, and the underlying mechanism may be related to the survival-enhancing microenvironment of the DA neurons.
Similar content being viewed by others
Introduction
Parkinson’s disease is a progressive, age-related movement disorder, which is also widely known as the frequent neurological disorder of the basal ganglia. Progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) further leads to the loss of dopaminergic neurotranmission in the striatum; following by a serious of profound functional changes of basal ganglia as well as voluntary movements1. Tremor, rigidity, akinesia and bradykinesia are considered as the main characteristics of movement disorders. Oxidative stress, apoptosis, neuroinflammation and mitochondrial dysfunction are the primary causes2. Recently, investigators have focused on the neurovascular unit (NVU), which is primarily composed of neurons and non-neuronal cells, including endothelial cells, pericytes, astrocytes, microglia and vascular smooth muscle cells. Glial cells physically play a neuroprotective role. Astrocytes envelop greater than 99% of the BBB endothelium, modulate the structure of endothelial cells and participate in the development of the BBB. In pathological condition, adverse stimulation causes glia to produce numerous neurotoxins and thereby contribute to the damage of neuroinflammation3,4. Activated microglia have been observed in postmortem SNpc tissues and PD models5. In a rat brain microvascular endothelial cells/microglia co-culture system, lipopolysaccharide (LPS) induces the activation of microglia, decreases the transendothelial electrical resistance (TEER), increases the paracellular transport of sodium fluorescein and damages the function of the BBB by producing reactive oxygen species6,7. TNF-α secreted by activated microglia increases the permeability of the BBB8, while proinflammatory cytokines, such as IL-1β, IL-4 and IFNγ, promote ICAM-1 expression to break down the normal structure of the NVU9. A cross-sectional analysis, comparing 38 normal elderly individuals with 100 PD patients revealed abnormal angiogenesis. Increased endothelial cells and blood vessels have been found in the SNpc area of PD patients and Parkinsonian primates10. BBB breakdown has also been detected11. Each component of the NVU seems to be closely connected and promote the progression of Parkinson’s disease. How to put off the procedure of neurodegeneration by regulating NVU is a thought-provoking question.
Pharmocological therapy has long been considered as the most efficient method for the treatment of PD. Existing treatments, like Madopar alleviate the motor symptoms but invalid in preventing neurodegeneration; dopamine agonist and MAO-B inhibitors may rise the side effects of abnormal motor output, so the study on supplemental herbs is quite essential12. The literature that shows the identification of patients with PD was first reported in 1,000 BC in India. The ancient civilizations used Mucuna pruriens to alleviate the symptoms. Recent years, natural extracts and monomers such as tannins, polyphenols, flavonoids, anthocyanins and terpenes have been extensively investigated in the medical sciences13.
Traditional Chinese medicine (TCM) has been used clinically for more than 3000 years. The Jia-Jian-Di-Huang-Yin-Zi decoction (JJDHYZ), which derives from a classical prescription in treating motor and language disorder, was effective in our previous clinical trials wherein both the Unified Parkinson’s Disease Rating Scale (UPDRS) scores and the Madopar dosage were reduced in PD patients14. Radix Rehmanniae, Fructus Corni, Radix Morindae Officinalis, Herba Cistanches, Radix Angelicae Sinensis, Radix Asparagi, Radix Paeoniae Alba are included in the prescription with a ratio of 1:0.6:1:1:1:1:1. And the main chemical compounds of these crude herbs are: Rubiadin (PubChem CID: 124062); Paeoniflorin (PubChem CID: 442534); Albiflorin (Pubchem CID: 24868421); Ferulic acid (Pubchem CID: 445858); Catalpol (Pubchem CID: 91520); Verbascose (Pubchem CID: 441434); Cistanoside D (PubChem CID: 5315930); Echinacoside (PubChem CID: 5281771); Rehmannioside A (PubChem CID: 6325881); Rehmannioside D (PubChem CID: 92044472) and Ursolic acid (PubChem CID: 64945). JJDHYZ showed behavior recovery and dopaminergic neurons protection by inhibiting apoptosis associated with mitochondrial and ER caspase 12 pathways in our previous study15. In the present study, we mimicked PD symptoms in a subacute mouse model using MPTP and investigated whether JJDHYZ played a regulatory role regarding DA neurons and their surrounding milieu.
Results
JJDHYZ-high dosage (JJDHYZ-H) improved the expression of glial-derived neurotrophic factor (GDNF) in MPTP-lesioned mice
In our previous study, JJDHYZ-H (34 g/kg/day) has been verified as the optimal dosage to ameliorate the behavior performance of PD mice, reduce the loss of DA neurons and restore the content of dopamine and its relevant metabolites15. To further clarify the neuroprotective role of JJDHYZ, we assessed the expressions of brain-derived neurotrophic factor (BDNF) and GDNF using immunofluorescence, real-time PCR and Western blot analysis. No significant differences were observed in the numbers of BDNF-positive cells, BDNF gene or protein expression among the groups (Fig. 1). However, MPTP significantly decreased the numbers of GDNF-positive cells (P < 0.01, Fig. 1A), GDNF mRNA and protein expression compared to that of the control group (P < 0.05, Fig. 1B,C). JJDHYZ-H and selegiline reversed the loss of GDNF-positive cells and expression compared to those of the MPTP-lesioned group (P < 0.01). Although JJDHYZ-L improved the levels of gene expression of GDNF (P < 0.05, Fig. 1C), no neuroprotective effect was detected using immunostaning and western blot (Fig. 1A,B).
JJDHYZ protected antioxidant enzyme activity
The activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) was measured. We also determined the concentration of malondialdehyde (MDA) to assess membrane lipid peroxidation. GSH (Fig. 2C) and CAT (Fig. 2D) were decreased significantly (P < 0.01) in the MPTP-lesioned mice, compared to the control animals. JJDHYZ-H and selegiline increased GSH activity and reduced MDA production compared to the model group (P < 0.05, Fig. 2A,C). No significant difference was observed in SOD (Fig. 2B). Neither JJDHYZ nor selegiline affected the rescuing activity of CAT after MPTP injection.
JJDHYZ-H inhibited the activation of microglia and astrocytes in the SN
We detected microglial and astrocyte activation in the substantia nigra of the control, model and JJDHYZ-H groups. GFAP was used as the biomarker of astrocyte, while Iba-1 and Tmem119 were used to mark the microglia. Few glial cells, indicators of neuroinflammation, were observed under normal, physical condition (A–C in Fig. 3a,c,e). However, numerous microglia and astrocytes were activated after MPTP administration in the model group (E-G). JJDHYZ-H invention inhibited the activation of microglia and astrocytes to some extent compared to those in the model group, although the results were still different from the control group (I-J).
JJDHYZ-H attenuated the ultrastructural changes in the BBB
The neuroprotective effects of JJDHYZ-H may potentially correlate with the changes in the BBB in subacute mouse models of PD. BBB ultrastructural alterations were detected using a transmission electron microscope (TEM). Microvessels in the SN of the control group were normal with a continuous basal lamina, astrocytic end-feet and normal endothelial cells (Fig. 4A). However, swollen endothelial cells and astrocytic end-feet, deflated capillary lumens and edema surrounding the microvessels were distinctly visible in the MPTP-lesioned group (Fig. 4B). JJDHYZ-H alleviated the swelling of the astrocytic end-feet. Microvessels with a continuous basal membrane and integrity of the endothelium were also observed (Fig. 4C).
JJDHYZ-H maintained claudin-5 and occludin expression and BBB permeability in the SNpc
We investigated the effect of JJDHYZ-H on two significant transmembrane tight junction proteins, claudin-5 and occludin. An immunostaining analysis of claudin-5 and occludin revealed changes in expression following the MPTP injection. A significant loss of claudin-5- and occludin-positive cells was found in the SNpc (P < 0.001, P < 0.05, respectively, Fig. 5B,C), and this loss was associated with an increase in BBB permeability (P < 0.05, Fig. 5E) in the MPTP group compared to that in the control group. JJDHYZ-H ameliorated the loss of both claudin-5 (P < 0.01) and occludin (P < 0.05), but no obvious changes in BBB permeability were observed using the Evans Blue method.
JJDHYZ-H reduced the abnormal increase in microvessels in the SNpc
CD31 was used to identify microvessels. We found that there were few CD31(+) microvessels in the SN of the control group (Fig. 6A). However, MPTP administration significantly increased the occurrence CD31(+) microvessels (Fig. 6B), and JJDHYZ-H reduced the number of abnormal microvessels compared to that in the model group (P < 0.05).
JJDHYZ-H down-regulated matrix metalloproteinase (MMP)2, MMP3 and MMP9 expression in the substantia nigra
MMP2, MMP3 and MMP9 were labeled via immunofluorescence (Fig. 7). The expression of all three of these MMPs was increased in the model group compared with that in the control group (P < 0.001) and the optimal JJDHYZ group (P < 0.01). Although down-regulation was observed, MMP3 and MMP9 expression was nevertheless different between the JJDHYZ-H and the control group (P < 0.05, Fig. 7D).
JJDHYZ-H regulated the expressions of IL-23, CCL2 and CCL4
Volcano plots are commonly used to analyze the mRNA expressions of microarray. Each point in the scatter plot represents a gene16. Based on published manuscripts and reviews, we listed the mostly studied 29 factors in Table 1, analyzed their gene expressions with PCR array and presented the result with volcano plots. The genes with fold-change (FC) >2 or <−2, besides p-value < 0.05 were considered as significant changed, and marked as the blue points out of the two perpendicular lines (Fig. 8A,B). Compared with the control group, MPTP model group down-regulated the expressions of CCL2, CCL4 and IL-23 as well as improved the expressions of CCL5, bFGF, NT-3 and Leukotrinens C4 (Fig. 8A). Similar to the control group, JJDHYZ-H reversed the inhibitory effects of MPTP on CCL2, CCL4 and IL-23; but it also up-regulated the other 8 cytokines’ expressions (Fig. 8B). Then the gene changes were processed with ingenuity pathway analysis (IPA). IPA maps molecules from the Ingenuity Pathway Knowledge Base. The computing methods of fold-changes of per gene were similar to the volcano plot. Statistical significant difference was assessed by Right-Tailed Fisher’s Exact Test17. The genetic interaction network (Fig. 8C,D) showed the molecular interactions under this experimental condition. In general, red color stands for the up-regulation of genes, green stands for the down-regulation. The more significant changed genes were marked with the darker colors. The lines and arrows annotated the types of molecular interactions. Interactions which have not been predicted are marked with the gray color. We verified the result with a multi-analyte ELISA array. Expressions of CCL2, CCL4 and IL-23 were significantly inhibited in the model group (P < 0.05), while no statistical difference were observed among the other cytokines or chemokines.
Discussion
Current treatments of PD mainly aim to alleviate clinical symptoms. As one of the potential intervening methods, neurotrophic factors play the roles of nourishing neurons, prompting axon regeneration18. The decrease of neurotrophic factors may induce the degeneration of dopaminergic neurons. Scientists immunostained the brain tissues of PD and non-PD patients aging 72–79 years to observe the expressions of various neurotrophic factors. Almost all the factors were decreased in some extend and the loss of GDNF was most outstanding19. Additionally, GDNF alleviated patients’ motor symptoms by putamen injection20, as well as promoted the survival of dopaminergic neurons in vitro21. In present study, we chose BDNF and GDNF to evaluate the neurotrophic effects of JJDHYZ decoction and observed that JJDHYZ in high dosage involved in up-regulating GDNF. Actually, the neuroprotective effects of JJDHYZ on PD mice were more than that.
Central nervous system is particularly sensitive to oxidative stress for its high oxygen consumption, high production of reactive oxygen and increasing deposition of neurotoxic metal irons22. Physically, antioxidant enzymes such as suoeroxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) catalyze free radicals. However, the auto-oxidation and enzymatic metabolism of dopamine leads to the production of neurotoxic free radicals and excessive oxygen species. Accumulated free radicals trigger oxidative stress to damage the biological molecules in cells and cause cell death23. Postmortem studies have revealed the reduction of GSH and increment of GSSG in the SNpc, which weaken the natural antioxidant defense and trigger the degeneration of nigral neurons24. In animal models, neurotoxin as MPTP induces the generation of free radicals and aggravated the burdens of antioxidant enzymes25. In our present study, we observed that MPTP inhibited the expressions of GSH, CAT and improved the concentration of MDA. JJDHYZ inhibited the generation of MDA, rescued the loss of the antioxidant GSH, especially at the high dosage.
With the in-depth study on PD, neuroinflammation is also considered to be associated with the selective loss of dopaminergic neurons. Therefore, the therapeutic intervene on microglia and astrocytes activation would be one of the effective strategies. Microglia are important immunity monitors that form the first defensive line against invading pathogens and rapidly respond to CNS injuries. Excessive microglia and astrocytes have been observed in the substantia nigra and striatum of autopsy cases26. Activated microglia and astrocytes generate ROS27 and release proinflammatory cytokines, chemokines and other proteinases28. The levels of IL-1β, IL-2, IL-6 and TNF-α are increased in the cerebrospinal fluid of patients with familial or sporadic PD29. Similar observations have been reported in MPTP- and 6-OHDA- induced animal models. These secreted factors promote alternations in the neuronal milieu and increase the neuronal susceptibility to death30,31. We double immunostained the GFAP with TH to detect activated astrocytes in the SN. In the meanwhile, we used two biomarkers to label microglia. Besides the well-known biomarker Iba-1, the other new cell-surface, microglia-specific marker named transmembrane protein 119 (Tmem119) was used32. The results demonstrated a significant loss of TH-positive cells concomitant with increased numbers of glial cells in the model group (Fig. 3). JJDHYZ-H treatment inhibited the loss of DA neurons and destructive neuroinflammation.
Through the results mentioned above, we primarily draw the conclusion that JJDHYZ had neuroprotective effects on PD mice. But the mechanisms were still uncertain. As the survival of dopaminergic neurons depends on the microenvironment, the potential connections between neurons and NVU attracted us most, so we further explored the regulated effects of JJDHYZ on NVU.
NVU is primarily composed of neurons and non-neuronal cells, including endothelial cells, pericytes, astrocytes, microglia and vascular smooth muscle cells. BBB is the key component. First, we observed the ultrastructural changes in the BBB by TEM. Normal microvessels were composed with continuous basal lamina, astrocytic end-feet and normal endothelial cells, which can be clearly observed under a transmission electron microscope. However, in MPTP-induced model, normal structures were instead by swollen endothelial cells and astrocytic end-feet, deflated capillary lumens and edema (Fig. 4B). JJDHYZ-H alleviated the swelling of the astrocytic end-feet. Microvessels with a continuous basal membrane and integrity of the endothelium were also observed (Fig. 4C). Angiogenesis is an important mechanism involved in the pathophysiology of PD33. Increased numbers of endothelial cell nuclei and blood vessels have been reported postmortem in the SNpc of patients with PD and prakinsonian primates. The increment of CD31 may related to the disruption of blood brain barrier and neuroinflammation11,34,35. For instance, MPTP-induced mice with TNF-α knockout were more possible to avoid the disruption of BBB36. Herein, we examined the BBB, neurons and glial cells in combination to deeply explore this treatment. Microvessels were labelled with an anti-CD31 antibody and more CD31-positive microvessels were found in the model group (Fig. 6, P < 0.05), which is in according with the reports of angiogenesis in PD33,35,37.
Microvessels regulate the exchanges of substances inside and outside the vessels and form a natural barrier to protect CNS. The tight junction proteins, which tightly connected are high selectivity to restrict the entrance of exogenous substances through the cell gap. We detected occludin and claudin-5 expression in the SNpc via immunostaining (Fig. 5). MPTP depleted these two tight junction proteins, while the proteins in the JJDHYZ-H-treated mice were not significantly different from those in the normal group. In addition to tight junctions, alternations of MMPs also influence the function of the BBB38. These zinc-dependent enzymes are key mediators of tight junction protein alterations, and exhibit proteolytic activity on the extracellular matrix, including the basal lamina in the NVU39. Under neuroinflammatory conditions, MMP3 and MMP9 are first secreted in response to inflammatory injury, and MMP2 is secreted in a zymogen form, which is activated upon host injury in the CNS40. All three of these MMPs cleave tight junction proteins. Occludin is vulnerable to attack by MMPs, MMP-2 and MMP-9 degrade claudin-5 after ischemic insult41. MMPs also degrade basal lamina proteins, such as fibronectin, laminin and heparan sulfate, after an ischemic insult, which contributes to BBB breakdown42,43. Several reports claimed that MMPs are involved in apoptosis. Dead DA neurons release MMP3 in vitro, which further induces microglial activation44. We fluorescently stained MMP2, MMP3 and MMP9, and determined that the expressions of all three of these MMPs was increased in the model group (Fig. 7). JJDHYZ-H maintained the expression of claudin-5 and occluding, probably by inhibiting the activation of MMPs.
To study the microenviromental changes of immune factors, we selected 29 factors from published articles, each of them was closely related with microglia and astrocytes. Compared to the control group, CCL2, CCL4 and IL-23 were down-regulated in the model group. JJDHYZ at high dosage reversed the inhibitory effects of MPTP on CCL2, CCL4 and IL-23 (Fig. 8). Increased CCL2-mRNA expression has been reported in an acute PD model induced by MPTP, and CCL2 may further induce the expression of CCL4; however, SNpc neuronal loss is not significant reduced in CCL2-knockout mice45. The role of CCL2 in PD is still uncertain. IL-23 is produced by myeloid cells and has been considered as one of the key cytokines for targeted therapies in immune-mediated diseases46,47. Based on former reports, CCL2 and CCL4 were thought to promote inflammation in the models of Alzheimer’s disease, while IL23 inhibits inflammation. Does this paradoxical with the neuroprotective effects of JJDHYZ? Since the role of these cytokines are unclear in PD, underlying mechanisms need to be investigated. The reason why JJDHYZ up-regulated CCL2,CCL4 and IL23 is still exploring and we will share the results in the future.
Materials and Methods
Ethics statement
The present study was performed according to the Guidelines for the Care and Use of Laboratory Animals from the National Institutes of Health. The Committee for Animal Research of Fudan University approved all procedures.
Animals
Male c57BL/6 mice weighing 22–25 g (aging 8–10 weeks) were housed in a temperature-controlled environment (constant temperature of 22 ± 3 °C) with 60% humidity and a 12-h light/dark cycle. Food and water were available ad libitum.
Preparation of JJDHYZ
The JJDHYZ decoction is composed of seven crude herbs: Radix Rehmanniae, Fructus Corni, Radix Morindae Officinalis, Herba Cistanches, Radix Angelicae Sinensis, Radix Asparagi and Radix Paeoniae Alba at a ratio of 1:0.6:1:1:1:1:1 (details about the herbs could be seen in Table 2). All the herbal components were purchased from Zhongshan Hospital, Fudan University. All crude herbs were first immerged in distilled water for 30 min and boiled for 1 h, and collected the suspension. Distilled water was then added into the herbal residues and the process was repeated twice. Dehydrated alcohol was added in the collected suspension to a final concentration of 75% alcohol (v/v). The residues were soaked in 75% ethyl alcohol for 24 h to collect the suspension. The suspension and liquid acquired from the residues were mixed, and centrifuged at 2,000 g for 20 min. The alcohol was completely volatilized by a rotary evaporator. The final concentration of the JJDHYZ decoction was 1 g/ml (w/v).
Chemical compounds of JJDHYZ
The JJDHYZ contains rubiadin (PubChem CID: 124062), paeoniflorin (PubChem CID: 442534), albiflorin (Pubchem CID: 24868421), ferulic acid (Pubchem CID: 445858), catalpol (Pubchem CID: 91520), verbascose (Pubchem CID: 441434), cistanoside D (PubChem CID: 5315930), echinacoside (PubChem CID: 5281771), rehmannioside A (PubChem CID: 6325881), rehmannioside D (PubChem CID: 92044472), and ursolic acid (PubChem CID: 64945).
Study design
C57BL/6 mice were orally administered saline, JJDHYZ or selegiline for 14 days followed by an intraperitoneal injection of saline or MPTP (30 mg/kg/day for 5 days). The mice were randomly divided into the following six groups: (1) control group: saline injection plus saline gastrointestinal administration; (2) MPTP group: MPTP injection plus saline; (3) JJDHYZ-low dosage group (JJDHYZ-L): MPTP injection plus JJDHYZ 8.5 g/kg/day; (4) JJDHYZ-middle dosage group (JJDHYZ-M): MPTP injection plus JJDHYZ 17 g/kg/day; (5) JJDHYZ-high dosage group (JJDHYZ-H): MPTP injection plus JJDHYZ 34 g/kg/day; and (6) selegiline group: MPTP injection plus selegiline 1.0 mg/kg/day48. MPTP powder and selegiline were solubilized in saline.
Immunohistochemistry
Sections of the SNpc (−3.64 mm~−2.92 mm from Bregma) were obtained from each mouse. The sections were dewaxed, hydrated, and washed with phosphate-buffered saline (PBS) (5 min × 3), and antigen retrieval was then performed in citric acid buffer (pH 6.0). Endogenous peroxidase activity was blocked with a 3% hydrogen peroxide-methanol buffer. Following incubation with 10% goat serum at room temperature for 1 h, the sections were incubated with an anti-occludin antibody (ab168986, Abcam) at 4 °C overnight. Subsequently, the immunolabeling was continued using a horseradish peroxide (HRP) secondary antibody (goat anti-rabbit, A0208, Beyotime). Immunopositive cells were counted from 5 slides of the SN region per mouse and the average was taken. 3 animals per group were used. All positive cell numbers were counted in SNpc under 40 magnification using the Image Pro Plus 6.0 software (Media Cybernetics, USA).
Immunofluorescence
Frozen sections of the SNpc were fixed in acetone for 20 min, treated with 3% hydrogen peroxide in methanol, and then incubated with a 10% goat serum blocking solution. Next, the sections were incubated at 4 °C overnight with antibodies against the following target proteins: BDNF (ab108319, Abcam), GDNF (ab18956, Abcam), Iba-1 (#019–19741, woko, Japan), Tmem119 (ab209064, Abcam), claudin-5 (ab15106, Abcam), MMP2 (ab92536, Abcam), MMP3 (ab53015, Abcam), and MMP9 (ab76003, Abcam). A Cy3-conjugated goat anti-rabbit antibody (A0516, Beyotime) was used as the secondary antibody. Nuclei were counterstained with DAPI (C1005, Beyotime). Images of the positively stained cells were captured using a fluorescence microscope (Olympus BX51, Japan). The quantification method was same as immunohistochemistry.
Quantitative RT-PCR analysis
Total RNA from SNpc tissues was extracted using the TRIzol reagent (T9424, Sigma) according to the manufacturer’s instructions. The concentration and quality of the mRNA were determined spectrophotometrically. Reverse-transcription reactions were performed using the PrimeScript RT reagent kit (RR047, Takara) on an Applied Biosystems 7500 Real-time PCR Detection System using a SYBR Premix Ex Taq II Kit (RR820, Takara). The following primers for GAPDH, GDNF, and BDNF were used: GAPDH: 5′-GGT TGT CTC CTG CGA CTT CA-3′ and 3′-CCT CAT TCT TTG GGA CCT GGT-5′; BDNF: 5′-GAG GTC TGA CGA CGA CAT CA-3′ and 3′-GTC AGT TCA CGG AA A CCT CG-5′; and GDNF:5′-CAA TGG ATT CAT ACC CTG-3′ and 3′-TCC AGA TAA TGT AGG TCG T-5′.All the samples were evaluated in triplicate, and averaged values were used for the 2−ΔΔCT relative quantification.
Western blot analysis
Western blot analysis was performed as reported previously49. The SNpc tissues were removed onto an ice-cold plate, lysed with a mixture containing RIPA and PMSF associated with phosphatase inhibitors, and then centrifuged at 13,500 rpm for 20 min at 4 °C. An equivalent amount of proteins was separated using SDS-PAGE gels and PVDF membranes. The membranes were blocked with 5% non-fat milk at room temperature for 1 h and then incubated overnight with primary antibodies against GDNF (1:1,000) and BDNF (1:1,000). Next, the membranes were incubated with a HRP-conjugated antibody against rabbit (dilution 1:1,000). The Immunoreactive bands were detected using enhanced chemiluminescence (P0018, Beyotime) and quantified using Image J software.
Enzyme-linked immunosorbent assay measurement
The lipid peroxidation end-product MDA (Catalog #K739, BioVision, USA) and the antioxidative enzymes SOD (Catalog#K335, BioVison), CAT (Catalog #K773, BioVision), and GSH (Catalog #K261, BioVision) were detected using ELISA kits according to the manufacturer’s instructions.
Ultrastructural alterations of the BBB
Mice were deeply anesthetized and then transcardially perfused with cold PBS. SNpc tissues were removed and divided into 1-mm3 segments. The samples were post fixed with 2.5% glutaraldehyde and 1% osmium for 2 h, followed by dehydration in a graded ethanol series and resin embedding. Ultrathin sections were sliced using a microtome, stained with 3% uranyl acetate-lead citrate and then observed with a TEM (PHILIPS CM-120).
Evaluation of BBB permeability
BBB permeability was evaluated by the leakage of Evans Blue (E2129, Sigma) into the brain after tail vein injection as described previously50. Briefly, 2% Evans Blue in normal saline was injected 1 h before the animals were euthanized. The mice were deeply anesthetized and transcardially perfused with normal saline. SNpc tissues were dissected and incubated in formamide (100 mg/ml) at 60 °C for 24 h, followed by centrifugation at 1,000 rpm for 5 min. The supernatants were collected, and the absorption of the tissues was detected using a luminescence spectrometer at a wavelength of 620 nm based on the standard curve of Evans Blue.
Statistical analysis
The data were analyzed using the SPSS 22.0 software employing a one-way analysis of variance (ANOVA) and using the post-hoc least significant difference (LSD) test. The results were expressed as the mean ± standard error for multiple group comparisons. Differences were considered statistically significant only when the P-value was less than 0.05 (P < 0.05).
References
Schmidt, N. & Ferger, B. Neurochemical findings in the MPTP model of Parkinson’s disease. Journal of Neural Transmission 108, 1263–1282, https://doi.org/10.1007/s007020100004 (2001).
Valente, M. E. et al. Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 304, 1158–1160, https://doi.org/10.1126/science.1096284 (2004).
Harish, G. et al. Characterization of traumatic brain injury in human brains reveals distinct cellular and molecular changes in contusion and pericontusion. Journal of neurochemistry 134, 156–172, https://doi.org/10.1111/jnc.13082 (2015).
Longhi, L. et al. Long-lasting protection in brain trauma by endotoxin preconditioning. Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 31, 1919–1929, https://doi.org/10.1038/jcbfm.2011.42 (2011).
Ponomarev, D. E., Veremeyko, T., Barteneva, N., Krichevsky, M. A. & Weiner, L. H. MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-alpha-PU.1 pathway. Nature medicine 17, 64–70, https://doi.org/10.1038/nm.2266 (2011).
Liebner, S., Kniesel, U., Kalbacher, H. & Wolburg, H. Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells. European journal of cell biology 79, 707–717, https://doi.org/10.1078/0171-9335-00101 (2000).
Rajasekaran, A. S., Beyenbach, W. K. & Rajasekaran, K. A. Interactions of tight junctions with membrane channels and transporters. Biochimica et biophysica acta 1778, 757–769, https://doi.org/10.1016/j.bbamem.2007.11.007 (2008).
Nishioku, T. et al. Tumor necrosis factor-alpha mediates the blood-brain barrier dysfunction induced by activated microglia in mouse brain microvascular endothelial cells. Journal of pharmacological sciences 112, 251–254, https://doi.org/10.2478/s13380-012-0027-8 (2010).
Huber, D. J., Campos, R. C., Mark, S. K. & Davis, P. T. Alterations in blood-brain barrier ICAM-1 expression and brain microglial activation after lambda-carrageenan-induced inflammatory pain. American journal of physiology. Heart and circulatory physiology 290, H732–740, https://doi.org/10.1152/ajpheart.00747.2005 (2006).
Barcia, C. et al. Changes in vascularization in substantia nigra pars compacta of monkeys rendered parkinsonian. Journal of neural transmission 112, 1237–1248, https://doi.org/10.1007/s00702-004-0256-2 (2005).
Gray, T. M. & Woulfe, M. J. Striatal blood-brain barrier permeability in Parkinson’s disease. Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 35, 747–750, https://doi.org/10.1038/jcbfm.2015.32 (2015).
Smulders, K. et al. Pharmacological treatment in Parkinson’s disease: effects on gait. Parkinsonism and related disorders 31, 3–13, https://doi.org/10.1016/j.parkreldis.2016.07.006 (2016).
da Costa, M. I. et al. Supplemental with herbal extracts to promote behavioral and neuroprotective effects in experimental models of Parkinson’s disease: a systematic review. Phytotherapy research 31, 959–970, https://doi.org/10.1002/ptr.5813 (2017).
Zhao, H., Li, W. W. & Gao, J. P. [Clinical trial on treatment of Parkinson’s disease of Gan-Shen yin deficiency type by recipe for nourishing Gan-Shen]. Zhongguo Zhong xi yi jie he za zhi (Zhongguo Zhongxiyi jiehe zazhi = Chinese journal of integrated traditional and Western medicine) 27, 780–784 (2007).
Zhang, J. et al. Jia-Jian-Di-Huang-Yin-Zi decoction reduces apoptosis induced by both mitochondrial and endoplasmic reticulum caspase12 pathways in the mouse model of Parkinson’s disease. Journal of ethnopharmacology 203, 69–79, https://doi.org/10.1016/j.jep.2016.12.053 (2017).
Li, W. et al. Using volcano plots and regularized-chi statistics in genetic association studies. Computational biology and chemistry 48, 77–83, https://doi.org/10.1016/j.compbiolchem.2013.02.003 (2014).
Racine, J. et al. comparison of genomic and proteomic data in recurrent airway obstruction affected horses using ingenuity pathway analysis. BMC veterinary research 7, 48, https://doi.org/10.1186/1746-6148-7-48 (2011).
Chauhan, B. N., Siegel, J. G. & Lee, M. J. Depletion of glial cell line-derived neurotrophic factor in substantia nigra neurons of Parkinson’s disease brain. Journal of chemical neuroanatomy 21, 277–288, https://doi.org/10.1016/S0891-0618(01)00115-6 (2001).
Gill, S. S. et al. Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson Disease. Nature medicine 9, 589–595, https://doi.org/10.1038/nm850 (2003).
Lang, E. A. et al. Randomized controlled trial of intraputamental glial cell line-derived neurotrophic factor infusion in Parkinson disease. Annals of neurology 59, 459–66, https://doi.org/10.1002/ana.20737 (2006).
Kumar, A. et al. GDNF overexpression from the native locus reveals its role in the nigrostriatal dopaminergic system function. PLOS genetic 11(12), e1005710, https://doi.org/10.1371/journal.pgen.1005710 (2015).
Chiurchiu, V., Orlacchio, A. & Maccarrone, M. Is modulation of oxidative stress an answer? The state of the art of redox therapeutic actions in neurodegenerative diseases. Oxidative medicine and cellular longevity 2016, 1–11, https://doi.org/10.1155/2016/7909380 (2016).
Magalingam, B. K., Radhakrishnan, K. A. & Haleagrahara, N. Protective mechanisms of flavonoids in Parkinson’s disease. Oxidative medicine and cellular longevity 2015, 1–14, https://doi.org/10.1155/2015/314560 (2015).
Gu, F., Chauhan, V. & Chauhan, A. Glutathione redox imbalance in brain disorders. Current opinion in clinical nutrition and metabolic care 18, 89–95, https://doi.org/10.1097/MCO.0000000000000134 (2015).
Marella, M., Seo, B. B., Yagi, T. & Matsuno-Yagi, A. Parkinson’s disease and mitochondrial complex I: a perspective on the Ndi1 therapy. Journal of bioenergetics and biomembranes 41, 493–497, https://doi.org/10.1007/s10863-009-9249-z (2009).
Santoro, M. et al. In-vivo evidence that high mobility group box 1 exerts deleterious effects in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model and Parkinson’s disease which can be attenuated by glycyrrhizin. Neurobiology of disease 91, 59–68, https://doi.org/10.1016/j.nbd.2016.02.018 (2016).
Sheng, S. W., Hu, S., Feng, A. & Rock, B. R. Reactive oxygen species from human astrocytes induced functional impairment and oxidative damage. Neurochemical research 38, 2148–2159, https://doi.org/10.1007/s11064-013-1123-z (2013).
Rezai-Zadeh, K., Gate, D. & Town, T. CNS infiltration of peripheral immune cells: D-Day for neurodegenerative disease? Journal of neuroimmune pharmacology: the official journal of the Society on NeuroImmune Pharmacology 4, 462–475, https://doi.org/10.1007/s11481-009-9166-2 (2009).
Brodacki, B. et al. Serum interleukin (IL-2, IL-10, IL-6, IL-4), TNFalpha, and INFgamma concentrations are elevated in patients with atypical and idiopathic parkinsonism. Neuroscience letters 441, 158–162, https://doi.org/10.1016/j.neulet.2008.06.040 (2008).
Ghosh, A. et al. Mitoapocynin Treatment Protects Against Neuroinflammation and Dopaminergic Neurodegeneration in a Preclinical Animal Model of Parkinson’s Disease. Journal of neuroimmune pharmacology: the official journal of the Society on NeuroImmune Pharmacology 11, 259–278, https://doi.org/10.1007/s11481-016-9650-4 (2016).
Nagatsu, T. & Sawada, M. Inflammatory process in Parkinson’s disease: role for cytokines. Current pharmaceutical design 11, 999–1016, https://doi.org/10.2174/1381612053381620 (2005).
Bennett, L. M. et al. New tools for studying microglia in the mouse and human CNS. PNAS 113(12), E1738–E1746, https://doi.org/10.1073/PNAS.1525528113 (2016).
Wada, K. et al. Expression levels of vascular endothelial growth factor and its receptors in Parkinson’s disease. Neuroreport 17, 705–709, https://doi.org/10.1097/01.wnr.0000215769.71657.65 (2006).
Carvey, M. P. et al. 6-Hydroxydopamine-induced alterations in blood-brain barrier permeability. The European journal of neuroscience 22, 1158–1168, https://doi.org/10.1111/j.1460-9568.2005.04281.x (2005).
Faucheux, A. B., Bonnet, M. A., Agid, Y. & Hirsch, C. E. Blood vessels change in the mesencephalon of patients with Parkinson’s disease. Lancet 353, 981–982, https://doi.org/10.1016/S0140-6736(99)00641-8 (1999).
Zhao, C., Ling, Z., Newman, B. M., Bhatia, A. & Carvey, M. P. TNF-alpha knockout and minocycline treatment attenuates blood-brain barrier leakage in MPTP-treated mice. Neurobiology of disease 26, 36–46, https://doi.org/10.1016/j.nbd.2006.11.012 (2007).
Janelidze, S. et al. Increased CSF biomarkers of angiogenesis in Parkinson disease. Neurology 85, 1834–1842, https://doi.org/10.1212/WNL.0000000000002151 (2015).
Gottschall, E. P. & Deb, S. Regulation of matrix metalloproteinase expressions in astrocytes, microglia and neurons. Neuroimmuno-modulation 3, 69–75, https://doi.org/10.1159/000097229 (1996).
Woessner, F. J. Jr. The family of matrix metalloproteinases. Annals of the New York Academy of Sciences 732, 11–21, https://doi.org/10.1111/j.1749-6632.1994.tb24720.x (1994).
Sirbulescu, F. R., Ilies, I. & Zupanc, K. G. Matrix metalloproteinase-2 and -9 in the cerebellum of teleost fish: Functional implications for adult neurogenesis. Molecular and cellular neurosciences 68, 9–23, https://doi.org/10.1016/j.mcn.2015.03.015 (2015).
Yang, T., Roder, E. K. & Abbruscato, J. T. Evaluation of bEnd5 cell line as an in vitro model for the blood-brain barrier under normal and hypoxic/aglycemic conditions. Journal of pharmaceutical sciences 96, 3196–3213, https://doi.org/10.1002/jps.21002 (2007).
Cheng, S., Pollock, S. A., Mahimkar, R., Olson, L. J. & Lovett, H. D. Matrix metalloproteinase 2 and basement membrane integrity: a unifying mechanism for progressive renal injury. FASEB journal: official publication of the Federation of American Societies for Experimental Biology 20, 1898–1900, https://doi.org/10.1096/fj.06-5898fje (2006).
Zlokovic, V. B. Remodeling after stroke. Nature medicine 12, 390–391, https://doi.org/10.1038/nm0406-390 (2006).
Kim, S. Y. et al. Matrix metalloproteinase-3: a novel signaling proteinase from apoptotic neuronal cells that activates microglia. The Journal of neuroscience: the official journal of the Society for Neuroscience 25, 3701–3711, https://doi.org/10.1523/JNEUROSCI.4346-04.2005 (2005).
Pattarini, R., Smeyne, J. R. & Morgan, I. J. Temporal mRNA profiles of inflammatory mediators in the murine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine model of Parkinson’s disease. Neuroscience 145, 654–668, https://doi.org/10.1016/j.neuroscience.2006.12.030 (2007).
Sheibanie, F. A., Tadmori, I., Jing, H., Vassiliou, E. & Ganea, D. Prostaglandin E2 induces IL-23 production in bone marrow-derived dendritic cells. FASEB journal: official publication of the Federation of American Societies for Experimental Biology 18, 1318–1320, https://doi.org/10.1096/fj.03-1367fje (2004).
Lyakh, L., Trinchieri, G., Provezza, L., Carra, G. & Gerosa, F. Regulation of interleukin-12/interleukin-23 production and the T-helper 17 response in humans. Immunological reviews 226, 112–131, https://doi.org/10.1111/j.1600-065X.2008.00700.x (2008).
Zhao, Q., Cai, D. & Bai, Y. Selegiline rescues gait deficits and the loss of dopaminergic neurons in a subacute MPTP mouse model of Parkinson’s disease. International journal of molecular medicine 32, 883–891, https://doi.org/10.3892/ijmm.2013.1450 (2013).
Zhang, J. et al. Neuroprotective Effects of Echinacoside on Regulating the Stress-Active p38MAPK and NF-kappaB p52 Signals in the Mice Model of Parkinson’s Disease. Neurochemical research 42, 975–985, https://doi.org/10.1007/s11064-016-2130-7 (2017).
Cao, G. et al. Ruscogenin Attenuates Cerebral Ischemia-Induced Blood-Brain Barrier Dysfunction by Suppressing TXNIP/NLRP3 Inflammasome Activation and the MAPK Pathway. International journal of molecular sciences 17, https://doi.org/10.3390/ijms17091418 (2016).
Acknowledgements
The work was supported by Grants from the National Natural Science Foundation of China (No. 81373866 & 81202813); the Three-year development plan project for Traditional Chinese Medicine (major research) of the Shanghai municipal health bureau (ZYSNXD-CC-ZDYJ028); the Three-year development plan project for Traditional Chinese Medicine (ZY3-CCCX-3-7003, ZY3-CCCX-1-1015); Outstanding youth fund of Zhongshan Hospital (2015ZSYXQN28).
Author information
Authors and Affiliations
Contributions
Jingsi Z., Jun X. and Dingfang C. designed the project. Jingsi Z., Zhennian Z.,Wen Z., Xiangting L., Ting W., Tingting L., Min C. and Zhonghai Y. performed experiments and analyzed the data. Jingsi Z. wrote the paper. All authors have reviewed the manuscript.
Corresponding authors
Ethics declarations
Competing Interests
The authors declare no competing interests.
Additional information
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Zhang, J., Zhang, Z., Zhang, W. et al. Jia-Jian-Di-Huang-Yin-Zi decoction exerts neuroprotective effects on dopaminergic neurons and their microenvironment. Sci Rep 8, 9886 (2018). https://doi.org/10.1038/s41598-018-27852-w
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-018-27852-w
This article is cited by
-
MSCs overexpressing GDNF restores brain structure and neurological function in rats with intracerebral hemorrhage
Molecular Biomedicine (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.