LEI 11718 PDF

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YL conducted animal behavior experiments and molecular biology experiments. FH conducted paper writing. JHC conducted molecular biology experiments and revised the paper. XQY conducted molecular biology experiments.

HZ designed the study and revised the paper. HYZ revised the preparation method of Chinese herbs and the composition of Houshiheisan.

QXZ and LW revised the paper. All authors approved the final version of the paper. A large number of randomized controlled trials have shown that Houshiheisan is effective in treating stroke, but its mechanism of action is unknown. Axonal remodeling is an important mechanism in neural protection and regeneration.

Therefore, this study explored the effect and mechanism of action of Houshiheisan on the repair of axons after cerebral ischemia. Rat models of focal cerebral ischemia were established by ligating the right middle cerebral artery. At 6 hours after model establishment, rats were intragastrically administered These medicines were intragastrically administered as above every 24 hours for 7 consecutive days.

Houshiheisanand its wind-dispelling and deficiency-nourishing ldi reduced the neurological deficit score and ameliorated axon and neuron lesions after cerebral ischemia. Furthermore, Houshiheisanand its wind-dispelling and deficiency-nourishing components decreased the expression of proteins that inhibit axonal remodeling: The effect of Houshiheisan was stronger than wind-dispelling drugs or leii drugs alone. These effects are strongest with Houshiheisan. Cerebral ischemia is a common neurological condition and leads to high rates of disability and mortality.

At present, the only approved therapy is human tissue-type plasminogen activator Lees et al.

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Moreover, even with effective thrombolysis, most patients will have varying degrees of neurological deficits Yamamoto et al. Stroke elicits disruption of myelin and impairs eli conductivity, which exacerbates functional outcome Benowitz and Carmichael, Axonal remodeling plays a crucial role in endogenous brain repair Zhang et al.

Some inhibitory factors of neurological regeneration, such as neurite outgrowth inhibitor protein A Nogo-Aplay an important role in restraining axon regeneration and lateral shoot formation Schwab, Also, low expression levels of axonal growth factors may be another difficulty for neural regeneration. Netrin-1 is 1718 important member of the netrin axonal guidance family, and is expressed in the mature central nervous system but not during development Izzi and Charron, The Netrin family proteins can promote the recovery of neuronal function after cerebral ischemia Bayat et al.

Furthermore, both promotive and inhibitory factors of axonal growth exert their functions by binding with their receptors and activating downstream molecular switches Sakumura et al. Based on these findings, exploring alternative therapies that boost axonal repair is a matter of urgency for stroke treatment.

Houshiheisan HSHS was the first prescription for stroke created by Zhang Zhongjing and has been clinically used in China to promote safe and effective repair of neurological function for over years. Our previous analysis of HSHS by high performance liquid chromatography showed that the main components are chlorogenic acid, apigeninO-glucoside, and luteolinO-glucoside Chang et al.


Importantly, chlorogenic acid ameliorates brain damage and edema by inhibiting matrix metalloproteinase-2 and 9 Lee et al. Furthermore, HSHS can inhibit amyloid precursor protein APP deposition, reduce the release of inflammatory response factors, and up-regulate the expression of brain-derived neurotrophic factor, thereby improving neurological function Bayat et al.

Our previous findings demonstrated that HSHS has leu potent effect on neural regeneration.

However, the mechanism of this effect has not been fully investigated. Seventy-two healthy, specific-pathogen-free, 3-month-old male Sprague-Dawley rats weighing — g, were provided by Beijing Vital River Laboratory Animal Technology Co.

Rats were anesthetized via face mask inhalation of 1. O 2 atmosphere and fixed in the supine position. The origins of the right common carotid artery, the internal carotid artery and the external carotid artery lie exposed and isolated by blunt leei. A monofilament nylon suture Beijing Sunbio Biotech Co.

Rats in the sham group were subjected to the same operation without insertion of the nylon suture. DND is composed of five Chinese herbs: Quality control of these Chinese herbs was previously performed by high performance liquid chromatography as published in our previous study Bayat et al. According to a previous study Bayat et al.

Drugs were intragastrically administrated for the first time at 6 hours after the operation, and then every 24 hours for 7 consecutive days. Neurological deficit was assessed using the improved Zea Longa five-grade scoring method Longa et al. Rats were trained from 3 days before the operation to ensure they could successfully cross a bar cm long, 3 cm wide and located 60 cm above the floor. The assessment was evaluated with the following grading criteria: Three rats were randomly selected from each of the five experimental groups at 7 days after modeling for hematoxylin-eosin staining.

After deep anesthesia, rats were transcardially perfused with mL 0. The ultrastructure of ischemic peri-infarcts in the hippocampus was observed by transmission electron microscopy JEM, Jeol, Japan.

Brains were then removed and ultrathin sections prepared. The ratio of axon diameter to total fiber diameter is called the g-ratio, and data were collected from 10 fields of view from each section and averaged using Image-Pro Plus 6. MAP-2 plays a critical role in axon growth Wang et al. Sections for immunofluorescence staining of MAP-2 were heated with 0. Sections were observed by fluorescence microscopy Olympus, Tokyo, Japanand images were collected by digital photomicroscopy Leica, Wetzlar, Germany.

Five fields of view were randomly selected from each section for quantitative analysis, and the average value of integral optical density calculated using the NIS-Elements Basic Research image acquisition system Nikon.

Total protein was extracted from ischemic hippocampi and quantified using a bicinchoninic acid protein assay kit. After several washes with Tris-buffered saline containing 0. One-way analysis of variance followed by the least significant difference post hoc test was utilized to compare mean difference among multiple groups.

Neurological function was evaluated by the Zea Longa five-level neurological deficit score every 24 hours for 7 days after pMCAO. C Hematoxylin-eosin staining of surviving neurons arrows in peri-infarct cortex upper and hippocampi lower at 7 days after cerebral ischemia original magnification: D Quantification of the number of surviving neurons. E Transmission electron microscopy images showing neurons with an irregular nuclear membrane arrow in peri-infarct hippocampi at 7 days original magnification: Permanent middle cerebral artery occlusion; WDD: The beam walking test was performed to observe fine motor control.


Hematoxylin-eosin staining revealed severe necrosis, infiltration of inflammatory cells and vascular edema in the cortex and hippocampus in the non-treatment pMCAO group.

Transmission electronic microscopy showed structural abnormalities of neurons following pMCAO. Pathological changes to myelin sheaths and axons were kei by transmission electron microscopy at 7 days after pMCAO Figure 3A. A Transmission electron microscopy images: Myelinated nerve fibers were disorganized; layers of the myelin sheath were wrinkled and axoplasms of axons were degenerated in the peri-infarct hippocampus arrow of the pMCAO group original magnification: B g-ratio of rats in each group.

C APP protein expression in peri-infarct hippocampi detected by western blot assays. The degree of axon damage was detected by western blot assay for APP.

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GAP is an indicator of axonal recovery. MAP-2 and GAP expression in the peri-infarct hippocampus of rats in each group at 7 days after cerebral ischemia. A GAP protein expression in peri-infarct hippocampi detected by western blotting. B Immunofluorescence staining of peri-infarct hippocampi showing MAP-2 positive neurons arrow. C Quantification of MAP-2 immunofluorescence staining. MAP-2 is another useful marker for evaluation of axonal recovery Roof et al. Nogo-A is a potent axonal inhibitory factor Schwab, A Nogo-A and NgR protein expression in peri-infarct hippocampi detected by western blotting.

B RhoA and Rock2 expression in peri-infarct hippocampi detected by western blotting. The expression of RhoA and Rock2 [a subtype of Rock with cardio-cerebral vascular distribution Iizuka et al. Netrin-1 promotes axonal remodeling by binding with its receptor, DCC Blasiak et al. A Netrin-1 and DCC protein expression in peri-infarct hippocampi detected by western blot assay.

B Rac1 and Cdc42 expression in peri-infarct hippocampi detected by western blot assay. Regeneration of neurons after cerebral ischemia, has been extensively investigated He et al. Neuroprotection plays an important role in neural regeneration after ischemic stroke Shi et al.

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HSHS and its components significantly alleviated ischemic injury in both neurons and axons. Importantly, HSHS and its components decreased the expression of APP, a neuronal transmembrane glycoprotein transported by rapid axonal transport, which rapidly accumulates after cerebral ischemia Coleman, Decreased levels of APP indicate less axon damage. HSHS promotes neuronal recovery after cerebral ischemia by up-regulating certain neurotrophic factors Bayat et al. Axonal remodeling is the main manifestation of neuronal recovery after cerebral ischemia Liu et al.

GAP, a growth-related protein promoting neuronal growth, development, nerve regeneration and synaptic remodeling, is a marker of axonal recovery after cerebral ischemia Li et al.

MAP-2 is another important molecule in regulating axonal reorganization Roof et al. However, DND did not produce a significant effect. 11781 recovery is regulated by guidance and inhibitory molecules Schmidt and Minnerup, ; Dun and Parkinson, However, axonal growth in adult neurons after cerebral 11178 is limited because of a lack of intrinsic capacity.

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