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Effect of Pei Yuan Tong Nao capsules on neuronal function and metabolism in cerebral ischemic rats
Journal of Ethnopharmacology 238 (2019) 111837
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Effect of Pei Yuan Tong Nao capsules on neuronal function and metabolism in cerebral ischemic rats
T
Jing Baia,b, Ying Gaoa,c,∗, Yong-Hong Gaoc a
Neurology Department, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China Beijing University of Chinese Medicine, China c Institute of Encephalopathy, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Pei Yuan Tong Nao Capsule Cerebral ischemia Ach α7nAchR Morris water maze
Ethnopharmacological significance: Pei Yuan Tong Nao (PYTN) capsules have been used in traditional Chinese medicine (TCM) for many centuries for the treatment of renal and vascular diseases, including cerebral ischemia. Although they are used in clinical practice, the evidence of their efficacy is lacking. Materials and methods: The cerebral ischemic rat model was established by bilateral carotid artery ligation and shear surgery. The laser speckle blood flow imaging apparatus was used to observe the changes in cerebral blood flow before and after surgery. The working memory of rats were analyzed using the Morris water maze (MWM) test. The 2-deoxy-2-[18F]fluoro-D-glucose (18-FDG) standard uptake values (SUVs) of rats with cerebral ischemia were evaluated by small-living animal positron emission tomography (PET) imaging. The quantity of acetylcholine in the hippocampus and the protein quantity of α7 nicotinic acetylcholine receptor in the hippocampus were detected with high-resolution fluid phase chromatography and Western blot analysis. Results: There was no significant difference in the preoperative mean cerebral blood perfusion between the groups. There was a significant decrease (P < 0.01) in cerebral perfusion in the model-operation, nimodipine, and PYTN groups after bilateral common carotid artery occlusion (BCCAo) compared with presurgery. The escape latency is shortened in the PYTN group (P < 0.05) compared with the model-operation group. SUVs of the nimodipine and PYTN groups increased (P < 0.05) compared with the model-operation group. After treatment with nimodipine and PYTN, acetylcholine levels in the hippocampus of the rats in the respective groups were lower than that of the sham-operation group (P < 0.01), and acetylcholine levels in the hippocampus of the nimodipine and PYTN groups were higher than that of the model-operation group (P < 0.05). α7 nicotinic acetylcholine receptor analysis showed that the protein levels of the nimodipine and PYTN groups increased after treatment compared with the model-operation group (P < 0.05). Conclusion: PYTN capsules improved the performance of rats with cerebral ischemia (analyzed using the MWM test), which may be due the to improvement of glucose metabolism in the hippocampus (evaluated by estimating acetylcholine and α7 nicotinic acetylcholine receptor protein).
1. Introduction Cerebrovascular diseases, including stroke, is the fifth leading cause of death worldwide. Stroke due to cerebral ischemia accounts for nearly 90% of all cases of stroke signifying the predominant pathologic mechanism behind cerebrovascular diseases in general and stroke in particular (Benjamin et al., 2017; Chen, 2012). The basic pathology of cerebral ischemia begins with mechanical occlusion of the cerebrovascular system due to a thrombus, which results in insufficient oxygen and glucose flow to the brain tissue supplied by the occluded artery. It could either be global brain ischemia or regional ischemia
∗
which depends on the occluded artery (Doyle et al., 2008). The resulting biochemical abnormalities lead to free radical production, ionic imbalance, inflammation, and apoptosis. Hence, the pathology of cerebral ischemia could be considered as a cascade culminating in tissue injury and cellular death (Krause et al., 1986; White et al., 2000). Therefore, targeting the biochemical cascade could minimize tissue injury (Chen, 2012). Further, the gross effects of cerebral ischemia affect cognitive function that impacts the quality of life in recuperating patients (Duncombe et al., 2017). The treatment option for cerebral ischemia in western medicine involves removal of the thrombus (thrombolytic therapy) by means of
Corresponding author. Neurology Department, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.Tel.: +86 13366275973. E-mail addresses: [email protected] (J. Bai), [email protected] (Y. Gao), [email protected] (Y.-H. Gao).
https://doi.org/10.1016/j.jep.2019.111837 Received 11 December 2018; Received in revised form 22 February 2019; Accepted 25 March 2019 Available online 26 March 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
Journal of Ethnopharmacology 238 (2019) 111837
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procedure, the cerebral blood flow and perfusion were determined by laser speckle contrast imaging (LASCA) technology, (PeriCam PSI System), which was facilitated by anesthetizing the animal with 10% chloral hydrate (3 mL/kg body weight) through intraperitoneal injection. After anesthetizing, the rats were placed on a white foam pad to maintain stable body temperature. The skin and fascial layers over the skull were retracted to expose the skull. The laser position was fixed on the middle by means of a P-3.0 suture. The software was set to detect the perfusion for 3 mm anterio-posteriorly and 4 mm latero-medially from the selected laser position. The cerebral blood flow was detected both before and after BCCAo. After determining normal cerebral blood flow, the rats were placed on the operating table in a crouching position. A sagittal dorsal midline incision was made and the fascia and muscles were separated along the medial edge of the sternocleidomastoid muscle. The right and left common carotid arteries were carefully separated from the respective vagus nerves and were gently stripped. Then the respective common carotid arteries were ligated and cut at both ends with a 6-0-sterilized wire harness. Finally, the neck incision was sutured. All the animals were intraperitoneally injected with 1 ml of penicillin injection to prevent postoperative infection.
tissue plasminogen activator (t-PA), which needs to be administered within 3 h of the initial symptoms. Apart from that, aspirin and anticoagulant-based preventive therapies are in use (Bednar Martin M. and Gross Cordell E., 1999; Hacke et al., 1995). Calcium channel blockers such as nimodipine have also been employed to alleviate neurological deficits in patients with ischemia (Gelmers and Hennerici, 1990). Traditional Chinese medicine (TCM) has been used in the treatment and prevention of ischemia throughout China (Han et al., 2017; Venketasubramanian et al., 2015). TCM regimen most often includes a mixed composition of herbs given in a specific ratio, which was finetuned by centuries-old empirical practice. As against the western medicinal practice of giving a drug for each indication, TCM has multiple roles, thereby eliminating the need to take multiple medications. Further, since most of the medicines are given as a combination of multiple drugs, the repertoire of the clinical indications for each drug combination also increases (Sucher, 2013, 2006). Different TCM herbs have anti-inflammatory and antioxidant properties, cause vasodilation, increase cerebral blood flow velocity, inhibit platelet aggregation, protect against reperfusion injury, and increase tissue tolerance to hypoxia (Sucher, 2006). According to TCM, a human being is considered as an integral whole connected with the surrounding world. The organs of the body are interconnected by the so called ‘’collaterals’’. Stroke and other vascular diseases including renal diseases belong to the “windrelated syndromes” that signifies movement of body fluids (blood, oxygen, metabolites, etc.). The different Chinese herbs that were evaluated in clinical trials for ischemic stroke include Huangqi injections, Naoan capsules, and Naomaitai capsules (Ma and Lan, 2011; Li-sha et al., 2013; Tan et al., 2013). Although other medicinal combination including Pei Yuan Tong Nao (PYTN) capsules have been used for the treatment of cerebral ischemia (Li, 2015), there are no preclinical study evaluating their efficacy. PYTN capsules consist of 14 different herbal ingredients that replenish the kidneys, which in TCM terminology means that they are potentially beneficial for any vascular “syndrome” including cerebral ischemia. At present, the treatment options for cerebral ischemia in western medicine are limited and scientific evidence of TCM for its treatment is scarce. Hence, in the current preclinical study, the efficacy of PYTN was evaluated in a rat model of cerebral ischemia. Nimodipine treatment was used as a comparator. The changes in acetylcholine levels served as a surrogate marker to analyze the biochemical parameters while the Morris water maze (MWM) test was used as an indicator to assess the gross cognitive improvement.
2.3. Experimental design The animals were divided into four groups: (1) sham-operated group (n = 8), in which the rats underwent the surgical procedure without common carotid artery ligation and occlusion to serve as a control for the surgical procedure; (2) experimental model-operation group (n = 8), in which the rats underwent BCCAo without subsequent treatment; (3) nimodipine group (n = 8), in which the rats subjected to BCCAo were treated with nimodipine as a standard of care comparator; and (4) PYTN group (n = 8), in which the rats subjected to BCCAo were treated with a standard dose of PYTN capsule. The animals were randomly assigned to the different study groups by means of random numbers generated by SPSS 24.0 statistical software. 2.4. PYTN capsule preparation A PYTN capsule consists of the following herbal and animal components: Fallopia multiflora (Thunb.) Haraldson (429 g), Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C.A. Mey (286 g), Asparagus cochinchinensis (Lour.) Merr. (286 g), vinegar-treated tortoise shell (Carapax Testudinis) (46 g), Cornu cervi pantotrichum (23 g), Cistanche deserticola Y.C. Ma (114 g), Cinnamomum cassia (L.) J. Presl (24 g), Radix Paeoniae Rubra (dried root of Paeonia lactiflora Pallas and Paeonia veitchii Lynch) (49 g), scorpion venom (Buthus martensii (Karsch)) (48 g), Hirudo nipponia Whitman (96 g), Lumbricus spp.(49 g), Crataegus pinnatifida Bunge (142 g), Poria cocos (Schw.) Wolf (48 g), and Glycyrrhizae radix (29 g). A dry extract powder containing all the above-mentioned herbs were provided by Henan Lingrui Pharmaceutical Co., Ltd (Peiyuantong brainstem extract powder, batch number 1709192). The chemical fingerprint was reaffirmed by highperformance liquid chromatography as part of internal quality assessment for each batch (data available with the author). Water was added to make a crude solution during the gavage. The PYTN dose administered was 0.486 g/kg, and in case of nimodipine, it was 8.1 mg/kg, which was based on Chen Qi's “Chinese Medicine Pharmacology Experimental Methodology” (Qi Chen, 1994). The prescribed doses were given through the intragastric route for the PYTN and nimodipine groups, while the corresponding amount of drinking water was administered for the sham-operation group. The animals were administered with the respective drugs once daily for 8 weeks after surgery.
2. Materials and Methods 2.1. Experimental animals Thirty-two healthy, white, male Sprague Dawley rats, weighing 250 g ± 10 g were purchased from Beijing Weitong Lihua experimental animal technology Co., Ltd., (license number SCXK (Beijing) 2012-0006). The animals were maintained in Dongzhimen Hospital, Beijing University, with free access to food and water, kept in a 12-h light-dark cycle mimicking natural environment. All the experimental procedures were approved by the Laboratory Animal Use Management Committee and the Ethics Committee (approval no. 2017-24) of the Dongzhimen Hospital. All the experimental procedures were done in accordance to ministry of cience and technology, People's Republic of China issued regulations and guidelines. The study was funded by Henan Lingrui Pharmaceutical Co., Ltd, grant number- HX-DZM2018009. 2.2. Surgical bilateral common carotid artery occlusion (BCCAo) model of cerebral ischemia
2.5. Behavioural testing using MWM The animal experimental model of cerebral ischemia was established by surgical occlusion of the carotid arteries. Before the surgical
The rats were trained for navigation through MWM for 8 weeks 2
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an Agilent 6410 QQQ LC-MS triple Quadrupole analysis system (California, United states), with the MassHunter Workstation.
before BCCAo. The working memory test was performed 2 days after BCCAo to check for cognitive performance. A day before the initiation of navigation training, the rats were allowed to swim for 3 min in the water maze without the platform to adapt to the environment. During navigation training, the rats were placed in the labyrinth from the midpoint of each quadrant, and the position of the platform remained unchanged (Section III). After reaching the platform in the respective quadrant, they were allowed to stay on the platform for 5 s. If the rats failed to find the platform within 60 s, they were guided to the platform and were allowed to stay for 5 s. The rats also received non-platform quadrant water training every day for a total of two block trainings (each block includes three trials). The interval between each trial was 60 s and the interval between the two blocks was 3 min for a total of 3 days of training during which the rats were taught to find the platform on their own. Spatial working memory: the assessment of trial-dependent learning was done 2 days after BCCAo. In the reference memory training stage, the platform was located at the North East (NE) position, whereas in the information coding stage (trial 1 of spatial working memory test), the platform position was changed to the diagonally opposite South West (SW) position. After 75 min of information coding stage (trial 1), the same trial was repeated to check the spatial memory retention (information extraction stage: trial 2) without the platform to calculate the time spent in the target quadrant zone. The memory parameters by MWM were analyzed by InterVideo 5.0 (Shanghai shift Information Technology Co., Ltd). The information coding and information extraction stages were compared with the incubation period (Vorhees and Williams, 2006; Xing et al., 2016; Zhu et al., 2014).
2.8. Estimation of α7 nicotinic acetylcholine receptor protein by Western blot analysis The tissue sample was weighed and 0.2 g of the brain tissue was mixed with 500 μL of RIPA lysate buffer containing protease and phosphatase inhibitors. After 30 min on ice, the homogenates were collected and were used as samples for Western blot analysis. Each lane contained a 50 μg of protein for purification in 10% Poly Acrylamide Gels. After wet transfer onto the polyvinylidene fluoride membrane, the blot was probed with Anti-Nicotinic Acetylcholine Receptor alpha 7 antibody (Abcam, ab10096) rabbit polyclonal antibody as the primary antibody and goat anti-rabbit-HRP antibody as the secondary antibody. 2.9. Statistical methods One-way analysis of variance and least significant difference test were performed by SPSS 24.0 statistical software for intragroup comparison. 3. Result 3.1. Cerebral blood perfusion changes before and after surgery There was no significant difference in preoperative cerebral blood flow perfusion between the groups. After BCCAo, the cerebral perfusion of the model-operation, nimodipine, and PYTN groups decreased significantly. There was no significant difference in the preoperative and postoperative cerebral blood perfusion in the sham-operation group confirming the successful establishment of the animal model with cerebral ischemia (Table 1 and Figs. 1 and 2).
2.6. Small-animal positron emission tomography in vivo imaging Positron emission tomography (PET) imaging was performed with the small-animal PET in vivo system (Inveon, Siemens). The computed tomography (CT) parameters were a tube voltage of 80 kV, a tube current of 500 μA, a total rotation of 220⁰, rotation steps of 120, an exposure time of 0.26 s, and a slice thickness of 0.2 mm. Prior to imaging, the rats were fasted for 6 h and were injected with 0.2. ml of 2deoxy-2-[18F]fluoro-D-glucose (18F-FDG) (500-1000u Ci) in the tail vein. The PET imaging parameters were set to emission acquisition mode, acquisition time of 45 min after the drug was administered, and PET collection time of 15 min, with a lateral field view of 7.8 cm and the axial field of 12.7 cm. The PET data were reconstructed using OSEM3D/MAP, and the reconstruction matrix was 128*128 with a resolution of 0.7 mm/pixel. Inveon Research Workplace software was used for data analysis. The SUVs for FDG were used to assess the response to therapy (nimodipine and PYTN). SUVs from the region of interest in each group of small animals were calculated with the following formula:
3.2. PYTN enhanced spatial memory acquisition post BCCAo The MWM test results (escape latency) in the information coding and information extraction stages were statistically compared between the following groups: Sham-operation vs model-operation; model-operation vs nimodipine; and model-operation vs PYTN and PYTN vs nimodipine.
SUV = radioactive concentration in brain (kBq/ml)/injected dose (MBq)/body weight (kg)
3.2.1. Information coding stage The escape latency in the information coding stage was compared between the sham-operation and the model-operation group to confirm the memory deficits caused by BCCAo. A statistically significant increase in escape latency was observed in the model-operation group compared with the sham-operation group in all the quadrants, except the NE suggesting that the BCCAo procedure impacted the cognitive memory functions (Table 2).
2.7. Detection of acetylcholine levels in the brain by liquid chromatographymass spectrometry
Table 1 Comparison of cerebral blood perfusion before and after surgery (mean ± SD, n = 8 in each group).
Hippocampus samples were weighed and 5 ml of physiological saline was added per gram of the weighed tissue to prepare it for homogenization. The mix was centrifuged at 5000 rpm for 10 min at 4 °C. To the mix, 200 μl of acetonitrile was added and centrifuged at 14,000 rpm for 10 min at 4 °C. Before injecting into the column, the supernatant was diluted five-folds with d9-brominated acetylcholine solution. The supernatant was loaded on to the solid-phase extraction columns (Zorbax Eclipse AAA, Shanghai, China). The analytes so obtained were dried by blowing hot air (300 °C). The dry extract was next taken for liquid chromatography-mass spectrometry (LC-MS) analysis in
Group
Pre- or post-surgery
Cerebral blood perfusion
Sham-operation
Presurgery Post-surgery Presurgery Post-surgery Presurgery Post-surgery Presurgery Post-surgery
187.96 183.17 180.97 122.63 177.41 128.33 187.08 133.99
Model-operation Nimodipine PYTN
± ± ± ± ± ± ± ±
36.66 40.23 27.55 11.62∗ 27.98 22.94∗ 34.83 32.18∗
* Statistically significant reduction in cerebral blood perfusion post-surgery (P < 0.01). 3
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Fig. 1. Comparison of intracranial vessels before and after 8 weeks of bilateral common carotid artery ligation- Real-time microcirculation imaging.
both PYTN- and nimodipine-treated animals showed a significant increase in acetylcholine levels compared with the model-operation group (0.25 ± 0.082 vs 0.131 ± 0.069; 0.23 ± 0.087 vs 0.131 ± 0.069). There was no significant difference in acetylcholine levels in PYTN-treated animals compared with nimodipine treatment (Table 5).
3.2.2. Information extraction stage A statistically significant increase in escape latency was observed for the model-operation group compared with the sham-operation group in all the quadrants. Statistically significant reductions in escape latency were also observed in the PYTN and nimodipine groups compared with the model-operation group, indicating that both the drugs were effective in enhancing the cognitive memory functions. There was no statistically significant difference between the PYTN group and the nimodipine group, suggesting that the PYTN capsule might be as effective as nimodipine in enhancing the memory functions (Table 3).
3.5. PYTN and nimodipine significantly increased relative gray value of α7 nicotinic acetylcholine receptor in the hippocampus Western blot analysis revealed that the α7-nicotinic acetylcholine receptor protein expression in the hippocampus was significantly lower in the model-operation group compared with the sham-operation group (0.28 ± 0.086 vs 0.53 ± 0.081) (Fig. 4). Further, both PYTN treatment and nimodipine treatment led to an increased expression of α7nicotinic acetylcholine receptor compared with the model-operation group (0.40 ± 0.095 vs 0.28 ± 0.086; 0.38 ± 0.082 vs 0.28 ± 0.086). This finding suggest that the enhanced cognitive memory function observed in PYTN-treated rats could be due to the increased neural activity as indicated by the acetylcholine and acetylcholine receptor levels.
3.3. PYTN and nimodipine increase SUVs for FDG by positron emission tomography-computed tomography imaging SUVs obtained by positron emission tomography-computed tomography imaging (PET-CT) scan revealed a significant reduction in the model-operation group compared with the sham-operation group, confirming that BCCAo has led to the observed reduction. A statistically significant increase in SUVs was also observed in the PYTN and nimodipine groups compared with the model-operation group indicative of positive response to therapy. Further, there was no statistically significant difference in PYTN compared to nimodipine, suggesting that PYTN capsules are as effective as nimodipine (Table 4 and Fig. 3).
4. Discussion
3.4. PYTN and nimodipine significantly increased acetylcholine levels in the hippocampus
Metabolic distress and subsequent cellular injury are salient features of hypoxic tissue. The neural damage that occurs during hypoperfusion due to cerebral ischemia is irreversible and the treatment options are very limited. Many TCM have been found to be effective in clinical practice but they lack the evidence-based medicine criteria set by western medicine. In this study, we demonstrated the enhancement in
HPLC revealed that there was a statistically significant decrease in acetylcholine levels in the model-operation group compared with the sham-operation group (0.131 ± 0.069 vs 0.277 ± 0.028). Further,
Fig. 2. Cerebral blood perfusion before (preoperative) and after (postoperative) BCCAo by laser speckle blood flow imaging. 4
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Table 2 Escape latency for each group in the information coding stage (mean ± SD, n = 8 in each group). Group
Platform quadrant (seconds)
Sham-operation Model-operation Nimodipine PYTN
SW
NW
SE
NE
16.985 ± 14.07 42.575 ± 15.05∗ 31.955 ± 17.61 26.75 ± 15.21
21.125 ± 8.22 38.75 ± 17.81∗ 32.375 ± 12.59 30.375 ± 7.65
17.625 30.125 24.625 25.485
± ± ± ±
7.6 9.42∗∗ 5.73 14.02
21.69 ± 9.49 32.125 ± 16.28 25.37 ± 14.27 23.45 ± 10.44
* The model-operation group compared with the sham-operation group (P < 0.05). ** The model-operation group compared with the sham-operation group (P < 0.01). Table 3 Multiple comparisons of escape latency for each group in the information extraction stage (mean ± SD, n = 8 in each group). Group
Platform quadrant (seconds)
Sham-operation Model-operation Nimodipine PYTN
SW
NW
10.405 ± 3.55 37.86 ± 4.76∗∗ 20.30 ± 4.46# 14.02 ± 3.26△△
13.78 27.14 14.73 15.53
SE ± ± ± ±
1.43 4.59∗ 2.43# 2.51△
8.725 26.06 15.53 11.73
NE ± ± ± ±
1.61 2.42∗∗ 2.51## 8.07△△
6.515 ± 1.44 23.23 ± 4.15∗∗ 14.61 ± 3.06 12.2 ± 2.37△
*P < 0.05 compared with sham-operation group. ** Compared with the sham-operation group P < 0.01. # P < 0.05 compared with the model-operation group. ## Compared with the model-operation group P < 0.01. △ Compared with the model-operation group P < 0.05. △△ Compared with the model-operation group P < 0.01.
evaluated by laser speckle blood flow imager. Sham-operation group, treatment-naïve group, and nimodipine standard of care groups were included to confirm the veracity of PYTN efficacy. The neural network in the hippocampus is responsible for spatial memory and hence we selected the MWM test that evaluates the reference and working spatial memory (Vorhees and Williams, 2006). The animals recalled the sample test and used the referral memory to travel in a shorter path to the platform in the second trial. When the platform quadrant was changed every day, the reference memory from the previous day could not be transferred to the next day. Hence, in the information extraction test, the memory recall of each day depends on the sample trial on the same day, measuring only temporary or working memory (Vorhees and Williams, 2006). Since acetylcholine and its receptor levels are a surrogate marker for active metabolism, we estimated them from hippocampal tissue. Our results indicate that there is active metabolism in PYTN- and
Table 4 SUVs of each group by PET-CT imaging (mean ± SD, n = 8 in each group).
SUV
Sham-operation
Model-operation
Nimodipine
PYTN
3.6 ± 0.36
2.20 ± 0.28∗∗
3.2 ± 0.28#
3.31 ± 0.31△
** Compared with the sham-operation group, significant reduction, P < 0.01. # Compared with the model-operation group, significant increase, P < 0.05. △ Compared with the model-operation group, significant increase, P < 0.05.
cognitive memory functions of PYTN capsules in chronic hypoperfused rats that mimic cerebral ischemia (Wakita et al., 1994). We further evaluated the metabolic activity of the affected tissues by estimating acetylcholine and its receptor levels in PYTN-treated, nimodipinetreated, and treatment-naïve rats. BCCAo blocked the supply of blood to the carotid system which was
Fig. 3. Representative PET-CT imaging of the four groups. 5
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Table 5 Acetylcholine and acetylcholine receptor levels in the hippocampus of each group (mean ± SD; n = 8, μg/g). Biological parameter
Mock-operation
Acetylcholine levels by HPLC Relative gray value of α7 nicotinic acetylcholine receptor by Western blot analysis
0.277 ± 0.028 0.53 ± 0.081
Model- operation ∗∗
0.131 ± 0.069 0.28 ± 0.086∗∗
Nimodipine
PYTN #
0.23 ± 0.087 0.38 ± 0.082#
0.25 ± 0.082△ 0.40 ± 0.095△△
** Compared with the sham-operation group, significant reduction, P < 0.01. ## Compared with the model-operation group, significant increase, P < 0.05. △ Compared with the model-operation group, significant increase, P < 0.05. △△ Compared with the model-operation group, significant increase, P < 0.01.
tissue. The potential limitations of the study include the shorter duration of observation which needs to be further confirmed in studies with longer follow-up. 5. Conclusion To conclude, we hereby provide preclinical evidence for the efficacy of PYTN capsules in the cerebral ischemic rat model which is as effective as nimodipine with potentially lower side effects. This could be further verified in well-planned clinical trials.
Fig. 4. Expression of α 7 acetylcholine receptor protein in the hippocampus by Western blot analysis.
Conflicts of interest nimodipine-treated animals, which suggests that the enhanced cognitive memory functions could be related to active metabolism. This was further confirmed by the observed SUVs in PET-CT scan. α 7 nicotinic acetylcholine receptor was used as potential therapeutic target in many other studies on cognitive disorders like Alzheimer's disease and schizophrenia. In case of cerebral ischemia, α 7 nicotinic acetylcholine receptor has been reported to reduce the macrophage production of pro-inflammatory cytokines thereby minimizing inflammation mediated tissue injury (Han et al., 2014). PYTN capsules have been used in TCM clinical practice for many years. According to the TCM principle, the kidneys are the most essential organs, and removing metabolic wastes helps in maintaining “blood stasis.” Since the kidneys are connected to other organs including the brain by means of “collaterals,” it is presumed that the beneficial effect of PYTN might be related to removal of toxic metabolites. The PYTN formulation consists of eight components of herbal origin, five components of animal origin, and one component of fungal origin. Some of the herbal components were also part of the traditional oriental medicine like Rehmannia glutinosa, which is also used in traditional Korean medicine for neurodegenerative disorders (Lee et al., 2011). In a previous study by Lee et al., Rehmannia glutinosa improved memory and reduced tissue damage by decreasing expression of proinflammatory cytokines in the hippocampus in scopolamine-induced memory impaired rats. They also observed that the decrease in inflammatory cytokine expression is mainly mediated by altering the cholinergic system (Lee et al., 2011). A previous study by Zhang et al. found catalpol to be the active component in Rehmannia glutinosa capable of neuroprotective function (Zhang et al., 2008). In a previous study, Cornu cervi pantotrichum, which is obtained from the antlers of sika deer, has been shown to minimize oxidative injury (Du and Cao, 2000). Similarly, Cistanche deserticola also possesses anti-oxidative properties that minimize reperfusion injury in ischemic tissues. Extracts of Cistanche deserticola rich in phenylethanoid glycoside has been shown to reduce myocardial ischemic reperfusion injury in a rat model. The same study also reported simultaneous upregulation of anti-apoptotic proteins and downregulation of cleaved caspase-3, suggesting that the precise mechanism might involve multiple pathways (Yu et al., 2016). At present, there is a scarcity of clinical evidence on the role of the other components of PYTN in medical literature. With the available evidence, we speculate that PYTN capsules enhance brain energy metabolism, thereby improving neuronal function in hypoperfused brain
The study was funded by Henan Lingrui Pharmaceutical Co., Ltd. The sponsors had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author contributions Jing Bai and Ying Gao conceived the idea, the experiments were performed by Jing Bai, Yong-Hong Gao and Ying Gao, and Jing Bai and Ying Gao wrote the manuscript. Funding The study was funded by Henan Lingrui Pharmaceutical Co., Ltd, grant number- HX-DZM-2018009. Acknowledgement The authors acknowledge Henan Lingrui Pharmaceutical Co., Ltd for the funding. List of abbreviations CT FDG HPLC MWM NE NW PET PYTN SE SD SUV SW TCM
computed tomography fluoro-D-glucose high-performance liquid chromatography Morris Water Maze North East North West positron emission tomography Pei Yuan Tong Nao South East Sprague Dawley standard uptake value South West traditional Chinese medicine
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Effect of Pei Yuan Tong Nao capsules on neuronal function and metabolism in cerebral ischemic rats
T
Jing Baia,b, Ying Gaoa,c,∗, Yong-Hong Gaoc a
Neurology Department, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China Beijing University of Chinese Medicine, China c Institute of Encephalopathy, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Pei Yuan Tong Nao Capsule Cerebral ischemia Ach α7nAchR Morris water maze
Ethnopharmacological significance: Pei Yuan Tong Nao (PYTN) capsules have been used in traditional Chinese medicine (TCM) for many centuries for the treatment of renal and vascular diseases, including cerebral ischemia. Although they are used in clinical practice, the evidence of their efficacy is lacking. Materials and methods: The cerebral ischemic rat model was established by bilateral carotid artery ligation and shear surgery. The laser speckle blood flow imaging apparatus was used to observe the changes in cerebral blood flow before and after surgery. The working memory of rats were analyzed using the Morris water maze (MWM) test. The 2-deoxy-2-[18F]fluoro-D-glucose (18-FDG) standard uptake values (SUVs) of rats with cerebral ischemia were evaluated by small-living animal positron emission tomography (PET) imaging. The quantity of acetylcholine in the hippocampus and the protein quantity of α7 nicotinic acetylcholine receptor in the hippocampus were detected with high-resolution fluid phase chromatography and Western blot analysis. Results: There was no significant difference in the preoperative mean cerebral blood perfusion between the groups. There was a significant decrease (P < 0.01) in cerebral perfusion in the model-operation, nimodipine, and PYTN groups after bilateral common carotid artery occlusion (BCCAo) compared with presurgery. The escape latency is shortened in the PYTN group (P < 0.05) compared with the model-operation group. SUVs of the nimodipine and PYTN groups increased (P < 0.05) compared with the model-operation group. After treatment with nimodipine and PYTN, acetylcholine levels in the hippocampus of the rats in the respective groups were lower than that of the sham-operation group (P < 0.01), and acetylcholine levels in the hippocampus of the nimodipine and PYTN groups were higher than that of the model-operation group (P < 0.05). α7 nicotinic acetylcholine receptor analysis showed that the protein levels of the nimodipine and PYTN groups increased after treatment compared with the model-operation group (P < 0.05). Conclusion: PYTN capsules improved the performance of rats with cerebral ischemia (analyzed using the MWM test), which may be due the to improvement of glucose metabolism in the hippocampus (evaluated by estimating acetylcholine and α7 nicotinic acetylcholine receptor protein).
1. Introduction Cerebrovascular diseases, including stroke, is the fifth leading cause of death worldwide. Stroke due to cerebral ischemia accounts for nearly 90% of all cases of stroke signifying the predominant pathologic mechanism behind cerebrovascular diseases in general and stroke in particular (Benjamin et al., 2017; Chen, 2012). The basic pathology of cerebral ischemia begins with mechanical occlusion of the cerebrovascular system due to a thrombus, which results in insufficient oxygen and glucose flow to the brain tissue supplied by the occluded artery. It could either be global brain ischemia or regional ischemia
∗
which depends on the occluded artery (Doyle et al., 2008). The resulting biochemical abnormalities lead to free radical production, ionic imbalance, inflammation, and apoptosis. Hence, the pathology of cerebral ischemia could be considered as a cascade culminating in tissue injury and cellular death (Krause et al., 1986; White et al., 2000). Therefore, targeting the biochemical cascade could minimize tissue injury (Chen, 2012). Further, the gross effects of cerebral ischemia affect cognitive function that impacts the quality of life in recuperating patients (Duncombe et al., 2017). The treatment option for cerebral ischemia in western medicine involves removal of the thrombus (thrombolytic therapy) by means of
Corresponding author. Neurology Department, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.Tel.: +86 13366275973. E-mail addresses: [email protected] (J. Bai), [email protected] (Y. Gao), [email protected] (Y.-H. Gao).
https://doi.org/10.1016/j.jep.2019.111837 Received 11 December 2018; Received in revised form 22 February 2019; Accepted 25 March 2019 Available online 26 March 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
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procedure, the cerebral blood flow and perfusion were determined by laser speckle contrast imaging (LASCA) technology, (PeriCam PSI System), which was facilitated by anesthetizing the animal with 10% chloral hydrate (3 mL/kg body weight) through intraperitoneal injection. After anesthetizing, the rats were placed on a white foam pad to maintain stable body temperature. The skin and fascial layers over the skull were retracted to expose the skull. The laser position was fixed on the middle by means of a P-3.0 suture. The software was set to detect the perfusion for 3 mm anterio-posteriorly and 4 mm latero-medially from the selected laser position. The cerebral blood flow was detected both before and after BCCAo. After determining normal cerebral blood flow, the rats were placed on the operating table in a crouching position. A sagittal dorsal midline incision was made and the fascia and muscles were separated along the medial edge of the sternocleidomastoid muscle. The right and left common carotid arteries were carefully separated from the respective vagus nerves and were gently stripped. Then the respective common carotid arteries were ligated and cut at both ends with a 6-0-sterilized wire harness. Finally, the neck incision was sutured. All the animals were intraperitoneally injected with 1 ml of penicillin injection to prevent postoperative infection.
tissue plasminogen activator (t-PA), which needs to be administered within 3 h of the initial symptoms. Apart from that, aspirin and anticoagulant-based preventive therapies are in use (Bednar Martin M. and Gross Cordell E., 1999; Hacke et al., 1995). Calcium channel blockers such as nimodipine have also been employed to alleviate neurological deficits in patients with ischemia (Gelmers and Hennerici, 1990). Traditional Chinese medicine (TCM) has been used in the treatment and prevention of ischemia throughout China (Han et al., 2017; Venketasubramanian et al., 2015). TCM regimen most often includes a mixed composition of herbs given in a specific ratio, which was finetuned by centuries-old empirical practice. As against the western medicinal practice of giving a drug for each indication, TCM has multiple roles, thereby eliminating the need to take multiple medications. Further, since most of the medicines are given as a combination of multiple drugs, the repertoire of the clinical indications for each drug combination also increases (Sucher, 2013, 2006). Different TCM herbs have anti-inflammatory and antioxidant properties, cause vasodilation, increase cerebral blood flow velocity, inhibit platelet aggregation, protect against reperfusion injury, and increase tissue tolerance to hypoxia (Sucher, 2006). According to TCM, a human being is considered as an integral whole connected with the surrounding world. The organs of the body are interconnected by the so called ‘’collaterals’’. Stroke and other vascular diseases including renal diseases belong to the “windrelated syndromes” that signifies movement of body fluids (blood, oxygen, metabolites, etc.). The different Chinese herbs that were evaluated in clinical trials for ischemic stroke include Huangqi injections, Naoan capsules, and Naomaitai capsules (Ma and Lan, 2011; Li-sha et al., 2013; Tan et al., 2013). Although other medicinal combination including Pei Yuan Tong Nao (PYTN) capsules have been used for the treatment of cerebral ischemia (Li, 2015), there are no preclinical study evaluating their efficacy. PYTN capsules consist of 14 different herbal ingredients that replenish the kidneys, which in TCM terminology means that they are potentially beneficial for any vascular “syndrome” including cerebral ischemia. At present, the treatment options for cerebral ischemia in western medicine are limited and scientific evidence of TCM for its treatment is scarce. Hence, in the current preclinical study, the efficacy of PYTN was evaluated in a rat model of cerebral ischemia. Nimodipine treatment was used as a comparator. The changes in acetylcholine levels served as a surrogate marker to analyze the biochemical parameters while the Morris water maze (MWM) test was used as an indicator to assess the gross cognitive improvement.
2.3. Experimental design The animals were divided into four groups: (1) sham-operated group (n = 8), in which the rats underwent the surgical procedure without common carotid artery ligation and occlusion to serve as a control for the surgical procedure; (2) experimental model-operation group (n = 8), in which the rats underwent BCCAo without subsequent treatment; (3) nimodipine group (n = 8), in which the rats subjected to BCCAo were treated with nimodipine as a standard of care comparator; and (4) PYTN group (n = 8), in which the rats subjected to BCCAo were treated with a standard dose of PYTN capsule. The animals were randomly assigned to the different study groups by means of random numbers generated by SPSS 24.0 statistical software. 2.4. PYTN capsule preparation A PYTN capsule consists of the following herbal and animal components: Fallopia multiflora (Thunb.) Haraldson (429 g), Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C.A. Mey (286 g), Asparagus cochinchinensis (Lour.) Merr. (286 g), vinegar-treated tortoise shell (Carapax Testudinis) (46 g), Cornu cervi pantotrichum (23 g), Cistanche deserticola Y.C. Ma (114 g), Cinnamomum cassia (L.) J. Presl (24 g), Radix Paeoniae Rubra (dried root of Paeonia lactiflora Pallas and Paeonia veitchii Lynch) (49 g), scorpion venom (Buthus martensii (Karsch)) (48 g), Hirudo nipponia Whitman (96 g), Lumbricus spp.(49 g), Crataegus pinnatifida Bunge (142 g), Poria cocos (Schw.) Wolf (48 g), and Glycyrrhizae radix (29 g). A dry extract powder containing all the above-mentioned herbs were provided by Henan Lingrui Pharmaceutical Co., Ltd (Peiyuantong brainstem extract powder, batch number 1709192). The chemical fingerprint was reaffirmed by highperformance liquid chromatography as part of internal quality assessment for each batch (data available with the author). Water was added to make a crude solution during the gavage. The PYTN dose administered was 0.486 g/kg, and in case of nimodipine, it was 8.1 mg/kg, which was based on Chen Qi's “Chinese Medicine Pharmacology Experimental Methodology” (Qi Chen, 1994). The prescribed doses were given through the intragastric route for the PYTN and nimodipine groups, while the corresponding amount of drinking water was administered for the sham-operation group. The animals were administered with the respective drugs once daily for 8 weeks after surgery.
2. Materials and Methods 2.1. Experimental animals Thirty-two healthy, white, male Sprague Dawley rats, weighing 250 g ± 10 g were purchased from Beijing Weitong Lihua experimental animal technology Co., Ltd., (license number SCXK (Beijing) 2012-0006). The animals were maintained in Dongzhimen Hospital, Beijing University, with free access to food and water, kept in a 12-h light-dark cycle mimicking natural environment. All the experimental procedures were approved by the Laboratory Animal Use Management Committee and the Ethics Committee (approval no. 2017-24) of the Dongzhimen Hospital. All the experimental procedures were done in accordance to ministry of cience and technology, People's Republic of China issued regulations and guidelines. The study was funded by Henan Lingrui Pharmaceutical Co., Ltd, grant number- HX-DZM2018009. 2.2. Surgical bilateral common carotid artery occlusion (BCCAo) model of cerebral ischemia
2.5. Behavioural testing using MWM The animal experimental model of cerebral ischemia was established by surgical occlusion of the carotid arteries. Before the surgical
The rats were trained for navigation through MWM for 8 weeks 2
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an Agilent 6410 QQQ LC-MS triple Quadrupole analysis system (California, United states), with the MassHunter Workstation.
before BCCAo. The working memory test was performed 2 days after BCCAo to check for cognitive performance. A day before the initiation of navigation training, the rats were allowed to swim for 3 min in the water maze without the platform to adapt to the environment. During navigation training, the rats were placed in the labyrinth from the midpoint of each quadrant, and the position of the platform remained unchanged (Section III). After reaching the platform in the respective quadrant, they were allowed to stay on the platform for 5 s. If the rats failed to find the platform within 60 s, they were guided to the platform and were allowed to stay for 5 s. The rats also received non-platform quadrant water training every day for a total of two block trainings (each block includes three trials). The interval between each trial was 60 s and the interval between the two blocks was 3 min for a total of 3 days of training during which the rats were taught to find the platform on their own. Spatial working memory: the assessment of trial-dependent learning was done 2 days after BCCAo. In the reference memory training stage, the platform was located at the North East (NE) position, whereas in the information coding stage (trial 1 of spatial working memory test), the platform position was changed to the diagonally opposite South West (SW) position. After 75 min of information coding stage (trial 1), the same trial was repeated to check the spatial memory retention (information extraction stage: trial 2) without the platform to calculate the time spent in the target quadrant zone. The memory parameters by MWM were analyzed by InterVideo 5.0 (Shanghai shift Information Technology Co., Ltd). The information coding and information extraction stages were compared with the incubation period (Vorhees and Williams, 2006; Xing et al., 2016; Zhu et al., 2014).
2.8. Estimation of α7 nicotinic acetylcholine receptor protein by Western blot analysis The tissue sample was weighed and 0.2 g of the brain tissue was mixed with 500 μL of RIPA lysate buffer containing protease and phosphatase inhibitors. After 30 min on ice, the homogenates were collected and were used as samples for Western blot analysis. Each lane contained a 50 μg of protein for purification in 10% Poly Acrylamide Gels. After wet transfer onto the polyvinylidene fluoride membrane, the blot was probed with Anti-Nicotinic Acetylcholine Receptor alpha 7 antibody (Abcam, ab10096) rabbit polyclonal antibody as the primary antibody and goat anti-rabbit-HRP antibody as the secondary antibody. 2.9. Statistical methods One-way analysis of variance and least significant difference test were performed by SPSS 24.0 statistical software for intragroup comparison. 3. Result 3.1. Cerebral blood perfusion changes before and after surgery There was no significant difference in preoperative cerebral blood flow perfusion between the groups. After BCCAo, the cerebral perfusion of the model-operation, nimodipine, and PYTN groups decreased significantly. There was no significant difference in the preoperative and postoperative cerebral blood perfusion in the sham-operation group confirming the successful establishment of the animal model with cerebral ischemia (Table 1 and Figs. 1 and 2).
2.6. Small-animal positron emission tomography in vivo imaging Positron emission tomography (PET) imaging was performed with the small-animal PET in vivo system (Inveon, Siemens). The computed tomography (CT) parameters were a tube voltage of 80 kV, a tube current of 500 μA, a total rotation of 220⁰, rotation steps of 120, an exposure time of 0.26 s, and a slice thickness of 0.2 mm. Prior to imaging, the rats were fasted for 6 h and were injected with 0.2. ml of 2deoxy-2-[18F]fluoro-D-glucose (18F-FDG) (500-1000u Ci) in the tail vein. The PET imaging parameters were set to emission acquisition mode, acquisition time of 45 min after the drug was administered, and PET collection time of 15 min, with a lateral field view of 7.8 cm and the axial field of 12.7 cm. The PET data were reconstructed using OSEM3D/MAP, and the reconstruction matrix was 128*128 with a resolution of 0.7 mm/pixel. Inveon Research Workplace software was used for data analysis. The SUVs for FDG were used to assess the response to therapy (nimodipine and PYTN). SUVs from the region of interest in each group of small animals were calculated with the following formula:
3.2. PYTN enhanced spatial memory acquisition post BCCAo The MWM test results (escape latency) in the information coding and information extraction stages were statistically compared between the following groups: Sham-operation vs model-operation; model-operation vs nimodipine; and model-operation vs PYTN and PYTN vs nimodipine.
SUV = radioactive concentration in brain (kBq/ml)/injected dose (MBq)/body weight (kg)
3.2.1. Information coding stage The escape latency in the information coding stage was compared between the sham-operation and the model-operation group to confirm the memory deficits caused by BCCAo. A statistically significant increase in escape latency was observed in the model-operation group compared with the sham-operation group in all the quadrants, except the NE suggesting that the BCCAo procedure impacted the cognitive memory functions (Table 2).
2.7. Detection of acetylcholine levels in the brain by liquid chromatographymass spectrometry
Table 1 Comparison of cerebral blood perfusion before and after surgery (mean ± SD, n = 8 in each group).
Hippocampus samples were weighed and 5 ml of physiological saline was added per gram of the weighed tissue to prepare it for homogenization. The mix was centrifuged at 5000 rpm for 10 min at 4 °C. To the mix, 200 μl of acetonitrile was added and centrifuged at 14,000 rpm for 10 min at 4 °C. Before injecting into the column, the supernatant was diluted five-folds with d9-brominated acetylcholine solution. The supernatant was loaded on to the solid-phase extraction columns (Zorbax Eclipse AAA, Shanghai, China). The analytes so obtained were dried by blowing hot air (300 °C). The dry extract was next taken for liquid chromatography-mass spectrometry (LC-MS) analysis in
Group
Pre- or post-surgery
Cerebral blood perfusion
Sham-operation
Presurgery Post-surgery Presurgery Post-surgery Presurgery Post-surgery Presurgery Post-surgery
187.96 183.17 180.97 122.63 177.41 128.33 187.08 133.99
Model-operation Nimodipine PYTN
± ± ± ± ± ± ± ±
36.66 40.23 27.55 11.62∗ 27.98 22.94∗ 34.83 32.18∗
* Statistically significant reduction in cerebral blood perfusion post-surgery (P < 0.01). 3
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Fig. 1. Comparison of intracranial vessels before and after 8 weeks of bilateral common carotid artery ligation- Real-time microcirculation imaging.
both PYTN- and nimodipine-treated animals showed a significant increase in acetylcholine levels compared with the model-operation group (0.25 ± 0.082 vs 0.131 ± 0.069; 0.23 ± 0.087 vs 0.131 ± 0.069). There was no significant difference in acetylcholine levels in PYTN-treated animals compared with nimodipine treatment (Table 5).
3.2.2. Information extraction stage A statistically significant increase in escape latency was observed for the model-operation group compared with the sham-operation group in all the quadrants. Statistically significant reductions in escape latency were also observed in the PYTN and nimodipine groups compared with the model-operation group, indicating that both the drugs were effective in enhancing the cognitive memory functions. There was no statistically significant difference between the PYTN group and the nimodipine group, suggesting that the PYTN capsule might be as effective as nimodipine in enhancing the memory functions (Table 3).
3.5. PYTN and nimodipine significantly increased relative gray value of α7 nicotinic acetylcholine receptor in the hippocampus Western blot analysis revealed that the α7-nicotinic acetylcholine receptor protein expression in the hippocampus was significantly lower in the model-operation group compared with the sham-operation group (0.28 ± 0.086 vs 0.53 ± 0.081) (Fig. 4). Further, both PYTN treatment and nimodipine treatment led to an increased expression of α7nicotinic acetylcholine receptor compared with the model-operation group (0.40 ± 0.095 vs 0.28 ± 0.086; 0.38 ± 0.082 vs 0.28 ± 0.086). This finding suggest that the enhanced cognitive memory function observed in PYTN-treated rats could be due to the increased neural activity as indicated by the acetylcholine and acetylcholine receptor levels.
3.3. PYTN and nimodipine increase SUVs for FDG by positron emission tomography-computed tomography imaging SUVs obtained by positron emission tomography-computed tomography imaging (PET-CT) scan revealed a significant reduction in the model-operation group compared with the sham-operation group, confirming that BCCAo has led to the observed reduction. A statistically significant increase in SUVs was also observed in the PYTN and nimodipine groups compared with the model-operation group indicative of positive response to therapy. Further, there was no statistically significant difference in PYTN compared to nimodipine, suggesting that PYTN capsules are as effective as nimodipine (Table 4 and Fig. 3).
4. Discussion
3.4. PYTN and nimodipine significantly increased acetylcholine levels in the hippocampus
Metabolic distress and subsequent cellular injury are salient features of hypoxic tissue. The neural damage that occurs during hypoperfusion due to cerebral ischemia is irreversible and the treatment options are very limited. Many TCM have been found to be effective in clinical practice but they lack the evidence-based medicine criteria set by western medicine. In this study, we demonstrated the enhancement in
HPLC revealed that there was a statistically significant decrease in acetylcholine levels in the model-operation group compared with the sham-operation group (0.131 ± 0.069 vs 0.277 ± 0.028). Further,
Fig. 2. Cerebral blood perfusion before (preoperative) and after (postoperative) BCCAo by laser speckle blood flow imaging. 4
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Table 2 Escape latency for each group in the information coding stage (mean ± SD, n = 8 in each group). Group
Platform quadrant (seconds)
Sham-operation Model-operation Nimodipine PYTN
SW
NW
SE
NE
16.985 ± 14.07 42.575 ± 15.05∗ 31.955 ± 17.61 26.75 ± 15.21
21.125 ± 8.22 38.75 ± 17.81∗ 32.375 ± 12.59 30.375 ± 7.65
17.625 30.125 24.625 25.485
± ± ± ±
7.6 9.42∗∗ 5.73 14.02
21.69 ± 9.49 32.125 ± 16.28 25.37 ± 14.27 23.45 ± 10.44
* The model-operation group compared with the sham-operation group (P < 0.05). ** The model-operation group compared with the sham-operation group (P < 0.01). Table 3 Multiple comparisons of escape latency for each group in the information extraction stage (mean ± SD, n = 8 in each group). Group
Platform quadrant (seconds)
Sham-operation Model-operation Nimodipine PYTN
SW
NW
10.405 ± 3.55 37.86 ± 4.76∗∗ 20.30 ± 4.46# 14.02 ± 3.26△△
13.78 27.14 14.73 15.53
SE ± ± ± ±
1.43 4.59∗ 2.43# 2.51△
8.725 26.06 15.53 11.73
NE ± ± ± ±
1.61 2.42∗∗ 2.51## 8.07△△
6.515 ± 1.44 23.23 ± 4.15∗∗ 14.61 ± 3.06 12.2 ± 2.37△
*P < 0.05 compared with sham-operation group. ** Compared with the sham-operation group P < 0.01. # P < 0.05 compared with the model-operation group. ## Compared with the model-operation group P < 0.01. △ Compared with the model-operation group P < 0.05. △△ Compared with the model-operation group P < 0.01.
evaluated by laser speckle blood flow imager. Sham-operation group, treatment-naïve group, and nimodipine standard of care groups were included to confirm the veracity of PYTN efficacy. The neural network in the hippocampus is responsible for spatial memory and hence we selected the MWM test that evaluates the reference and working spatial memory (Vorhees and Williams, 2006). The animals recalled the sample test and used the referral memory to travel in a shorter path to the platform in the second trial. When the platform quadrant was changed every day, the reference memory from the previous day could not be transferred to the next day. Hence, in the information extraction test, the memory recall of each day depends on the sample trial on the same day, measuring only temporary or working memory (Vorhees and Williams, 2006). Since acetylcholine and its receptor levels are a surrogate marker for active metabolism, we estimated them from hippocampal tissue. Our results indicate that there is active metabolism in PYTN- and
Table 4 SUVs of each group by PET-CT imaging (mean ± SD, n = 8 in each group).
SUV
Sham-operation
Model-operation
Nimodipine
PYTN
3.6 ± 0.36
2.20 ± 0.28∗∗
3.2 ± 0.28#
3.31 ± 0.31△
** Compared with the sham-operation group, significant reduction, P < 0.01. # Compared with the model-operation group, significant increase, P < 0.05. △ Compared with the model-operation group, significant increase, P < 0.05.
cognitive memory functions of PYTN capsules in chronic hypoperfused rats that mimic cerebral ischemia (Wakita et al., 1994). We further evaluated the metabolic activity of the affected tissues by estimating acetylcholine and its receptor levels in PYTN-treated, nimodipinetreated, and treatment-naïve rats. BCCAo blocked the supply of blood to the carotid system which was
Fig. 3. Representative PET-CT imaging of the four groups. 5
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J. Bai, et al.
Table 5 Acetylcholine and acetylcholine receptor levels in the hippocampus of each group (mean ± SD; n = 8, μg/g). Biological parameter
Mock-operation
Acetylcholine levels by HPLC Relative gray value of α7 nicotinic acetylcholine receptor by Western blot analysis
0.277 ± 0.028 0.53 ± 0.081
Model- operation ∗∗
0.131 ± 0.069 0.28 ± 0.086∗∗
Nimodipine
PYTN #
0.23 ± 0.087 0.38 ± 0.082#
0.25 ± 0.082△ 0.40 ± 0.095△△
** Compared with the sham-operation group, significant reduction, P < 0.01. ## Compared with the model-operation group, significant increase, P < 0.05. △ Compared with the model-operation group, significant increase, P < 0.05. △△ Compared with the model-operation group, significant increase, P < 0.01.
tissue. The potential limitations of the study include the shorter duration of observation which needs to be further confirmed in studies with longer follow-up. 5. Conclusion To conclude, we hereby provide preclinical evidence for the efficacy of PYTN capsules in the cerebral ischemic rat model which is as effective as nimodipine with potentially lower side effects. This could be further verified in well-planned clinical trials.
Fig. 4. Expression of α 7 acetylcholine receptor protein in the hippocampus by Western blot analysis.
Conflicts of interest nimodipine-treated animals, which suggests that the enhanced cognitive memory functions could be related to active metabolism. This was further confirmed by the observed SUVs in PET-CT scan. α 7 nicotinic acetylcholine receptor was used as potential therapeutic target in many other studies on cognitive disorders like Alzheimer's disease and schizophrenia. In case of cerebral ischemia, α 7 nicotinic acetylcholine receptor has been reported to reduce the macrophage production of pro-inflammatory cytokines thereby minimizing inflammation mediated tissue injury (Han et al., 2014). PYTN capsules have been used in TCM clinical practice for many years. According to the TCM principle, the kidneys are the most essential organs, and removing metabolic wastes helps in maintaining “blood stasis.” Since the kidneys are connected to other organs including the brain by means of “collaterals,” it is presumed that the beneficial effect of PYTN might be related to removal of toxic metabolites. The PYTN formulation consists of eight components of herbal origin, five components of animal origin, and one component of fungal origin. Some of the herbal components were also part of the traditional oriental medicine like Rehmannia glutinosa, which is also used in traditional Korean medicine for neurodegenerative disorders (Lee et al., 2011). In a previous study by Lee et al., Rehmannia glutinosa improved memory and reduced tissue damage by decreasing expression of proinflammatory cytokines in the hippocampus in scopolamine-induced memory impaired rats. They also observed that the decrease in inflammatory cytokine expression is mainly mediated by altering the cholinergic system (Lee et al., 2011). A previous study by Zhang et al. found catalpol to be the active component in Rehmannia glutinosa capable of neuroprotective function (Zhang et al., 2008). In a previous study, Cornu cervi pantotrichum, which is obtained from the antlers of sika deer, has been shown to minimize oxidative injury (Du and Cao, 2000). Similarly, Cistanche deserticola also possesses anti-oxidative properties that minimize reperfusion injury in ischemic tissues. Extracts of Cistanche deserticola rich in phenylethanoid glycoside has been shown to reduce myocardial ischemic reperfusion injury in a rat model. The same study also reported simultaneous upregulation of anti-apoptotic proteins and downregulation of cleaved caspase-3, suggesting that the precise mechanism might involve multiple pathways (Yu et al., 2016). At present, there is a scarcity of clinical evidence on the role of the other components of PYTN in medical literature. With the available evidence, we speculate that PYTN capsules enhance brain energy metabolism, thereby improving neuronal function in hypoperfused brain
The study was funded by Henan Lingrui Pharmaceutical Co., Ltd. The sponsors had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author contributions Jing Bai and Ying Gao conceived the idea, the experiments were performed by Jing Bai, Yong-Hong Gao and Ying Gao, and Jing Bai and Ying Gao wrote the manuscript. Funding The study was funded by Henan Lingrui Pharmaceutical Co., Ltd, grant number- HX-DZM-2018009. Acknowledgement The authors acknowledge Henan Lingrui Pharmaceutical Co., Ltd for the funding. List of abbreviations CT FDG HPLC MWM NE NW PET PYTN SE SD SUV SW TCM
computed tomography fluoro-D-glucose high-performance liquid chromatography Morris Water Maze North East North West positron emission tomography Pei Yuan Tong Nao South East Sprague Dawley standard uptake value South West traditional Chinese medicine
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