Part Two | Jia-Jian-Di-Huang-Yin-Zi Decoction Exerts Neuroprotective Effects On Dopaminergic Neurons And Their Microenvironment

Part Two | How does Chinese herbal medicine such as Cistanche play a neuroprotective effect and improve memory?

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Discussion

Current treatments of PD mainly aim to alleviate clinical symptoms. As one of the potential intervening methods, neurotrophic factors play the role of nourishing neurons, prompting axon regeneration18. Te decrease of neurotrophic factors may induce the degeneration of dopaminergic neurons. Scientists immunostained the brain tissues of PD and non-PD patients aged 72–79 years to observe the expressions of various neurotrophic factors. Almost all the factors were decreased to some extent 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 the present study, we chose BDNF and GDNF to evaluate the neurotrophic effects of JJDHYZ decoction and observed that JJDHYZ in high dosage is involved in up-regulating GDNF. Actually, the neuroprotective effects of JJDHYZ on PD mice were more than that. The 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 superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) catalyze free radicals. However, the auto-oxidation and enzymatic metabolism of dopamine lead 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 aggravate 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. Te 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 the continuous basal lamina, astrocytic end-feet, and normal endothelial cells, which can be clearly observed under a transmission electron microscope. However, in the MPTP-induced model, normal structures were instead by swollen endothelial cells and astrocytic end-feet, defeated 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 parkinsonian primates. The increment of CD31 may be related to the disruption of the blood-brain barrier and neuroinfammation11,34,35. 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 labeled with an anti-CD31 antibody and more CD31-positive microvessels were found in the model group (Fig. 6, P<0.05), which is in accordance with the reports of angiogenesis in PD.

Microvessels regulate the exchanges of substances inside and outside the vessels and form a natural barrier to protect CNS. The tight junction proteins, which are 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. Tese 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 were 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 microenvironmental changes of immune factors, we selected 29 factors from published articles, each of which was closely related to 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 significantly 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 is 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.

Te JJDHYZ decoction is composed of seven crude herbs: Radix Rehmanniae, Fructus Corni, Radix Morinda 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 immersed in distilled water for 30min and boiled for 1h and collected the suspension. Distilled water was then added to the herbal residues and the process was repeated twice. Dehydrated alcohol was added to 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.

Te JJDHYZ contains rubiadin (PubChem CID: 124062), paeoniforin (PubChem CID: 442534), albiforin (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 (30mg/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 1.0mg/kg/day48. MPTP powder and selegiline were solubilized in saline.

Immunohistochemistry.

Sections of the SNpc (−3.64mm~−2.92mm from Bregma) were obtained from each mouse. The sections were dewaxed, hydrated, and washed with phosphate-buffered saline (PBS) (5min×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 1h, 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 20min, 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, woke, 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 the 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 an 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. Te 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 an HRP-conjugated antibody against rabbits (dilution 1:1,000). Te 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 2h, 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 1h before the animals were euthanized. Te mice were deeply anesthetized and transcardially perfused with normal saline. SNpc tissues were dissected and incubated in formamide (100mg/ml) at 60 °C for 24h, followed by centrifugation at 1,000 rpm for 5 min. Te supernatants were collected, and the absorption of the tissues was detected using a luminescence spectrometer at a wavelength of 620nm 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 hocleast 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).