- Email: [email protected]
Compound K attenuated hepatectomy-induced post-operative cognitive dysfunction in aged mice via LXRα activation
Biomedicine & Pharmacotherapy 119 (2019) 109400
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Compound K attenuated hepatectomy-induced post-operative cognitive dysfunction in aged mice via LXRα activation
T
Qifang Liua, Lidan Liub, Hongmei Liuc, Jingjing Jiangb, Shanbin Guod, Cong Wangb, Yi Jiac, ⁎ Yue Tianb, a
Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang, 110004, China Department of Anesthesiology, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang, 110004, China c Institute of Materia Medica and Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, 30 Gaotanyan Street, Chongqing, 400038, China d Department of Pharmacy, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang, 110004, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Compound K Inflammation Liver X receptor Post-operative cognitive dysfunction
Aims: Post-operative cognitive dysfunction (POCD) occurs after major surgery in elderly patients and affects the quality of patients’ lives. The present study aims to explore the protective effects and possible mechanisms of compound K in old mice with POCD caused by partial hepatectomy. Methods: Sixteen month-old mice were administered different doses of compound K from the 8th day to 14th day after partial hepatectomy. Cognitive function was subsequently measured with a Morris water-maze (MWM) test. Serum inflammatory cytokine levels were measured by magnetic bead panel; levels of cytokines in the hippocampus were analyzed using immunohistochemistry and immunoblotting. The mRNA levels of target genes were measured using real-time PCR. Results: Compared with the model group, MWM scores were significantly attenuated at days 10 and 14 postsurgery in mice receiving compound K (10, 30 mg/kg) in a dose-dependent manner. Both systemic and local cytokine levels were reduced after treatment of compound K. The alterations in serum lipids were independent of the attenuation of POCD syndrome. An inhibitor of liver X receptor-α (LXRα), GGPP, reversed the effects of compound K. Conclusions: The results provide evidence for an alleviation of POCD by compound K via local inflammation inhibition in hippocampus tissue; furthermore, the data suggests the mechanism involves the LXRα pathway. The present study supports further evaluation of compound K as a potential effective modulator for POCD.
1. Introduction Post-operative cognitive dysfunction (POCD), which is defined by various alterations of a patient’s cognitive functioning compared to their pre-surgery status, is one of the cognitive disorders found in the postoperative period [1]. POCD occurs frequently after patients undergo certain major surgical operations, especially in the elderly, and seriously affects patients’ quality of life [2]. The pathophysiology and possible mechanism of POCD is less well defined. Hyperventilation [3], hypotension [4], cerebral microemboli [5], and inflammation [6] are thought to be involved in the process of POCD. As indicated in many previous studies, systemic and cerebral inflammatory reactions [7], especially the activation of the NF-κB signaling pathway [8,9], play a major role in POCD. Hence the regulation of inflammation might be a potential effective therapeutic target for POCD, in addition to other
⁎
symptomatic treatments. Panax notoginseng (Burk.) F. H. Chen has been widely used as a major natural remedy in traditional medicine for the recovery of trauma for about 1000 years in Asia. Our previous studies have showed that one of the dammarane-type triterpene active metabolic components of Panax notoginseng saponins, 20-O-d-glucopyranosyl-20(S)-protopanaxadiol (compound K, 10 and 30 mg/kg in mice), can reduce inflammatory reactions in atherosclerosis lesions via liver X receptor α (LXRα) activation [10]. In addition, multiple other pharmaceutical activities have been reported for compound K, including anti-arthritis [11,12], anti-tumor [13,14], anti-diabetes [15,16], skin protection [17], neuroprotection [18] and anti-inflammation [19,20]. Recent studies have clearly shown the importance of the LXR pathway in both the inflammatory reaction and general immunological regulation. For example, GW3965, an LXR agonist, has been shown to alleviate the
Corresponding author. E-mail address: [email protected] (Y. Tian).
https://doi.org/10.1016/j.biopha.2019.109400 Received 13 June 2019; Received in revised form 22 August 2019; Accepted 28 August 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
Fig. 1. Compound K (CK) improved Morris water-maze (MWM) test scores of POCD mice. A. The administration of compound K (10, 30 mg/kg) reduced the escape latency (time until reaching the platform, day 10 and 14) as compared to the model group. B. The administration of compound K (10, 30 mg/kg) increased the time spent in the original platform quadrant as compared with the model group (day 11 and 15). C. Treatment with compound K (10, 30 mg/kg) increased the total number of crossings of the original platform quadrant of mice as compared with the model group (day 11 and 15). *p < 0.05. Data are presented as the mean ± SD. n = 8.
compound K acts on POCD. Towards this end, we evaluated compound K using an animal model of POCD induced by partial hepatectomy in old mice, as we have previously reported [25].
inflammation reaction in brain [21], adipose tissue [22] and atherosclerosis lesions [23], independent with the activation of glucocorticoid receptor [24]. Therefore, the present study was undertaken to further understand the protective effect and underlying mechanisms by which 2
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
Fig. 2. Effects of compound K (CK) on serum glucose, weight, serum inflammatory cytokines, and lipids levels in aged mice post-surgery. A. There was no significant difference of serum glucose levels among the groups. B. There was no significant difference of body weights among the groups. C. Serum inflammatory cytokines (TNF-α and IL-1β) in the model group were increased significantly as compared with the control group; furthermore, treatment with compound K (10, 30 mg/kg) significantly decreased levels of inflammatory cytokines as compared with the model group. D. There was no significant difference in serum triglyceride or total cholesterol levels among the groups. The administration of compound K (10, 30 mg/kg) significantly decreased LDL levels and increased HDL levels as compared with the model group. *p < 0.05. Data are presented as the mean ± SD. n=8.
2. Methods
China Medical University. All animals underwent a common one-week acclimatization period and were housed singly in a temperature-controlled (22–25 °C) environment on a 12 h light-dark cycle, under specific pathogen-free conditions, with food and water given ad libitum. The body weight of each animal was monitored every three days. To explore the potential effects of compound K on POCD, the mice were randomly divided into seven groups (n = 8). The mice in model group underwent partial hepatectomy under anesthesia, while the animals in control group were underwent a sham operation (same protocol as model group, expect liver excision). Mice in vehicle and compound K groups were administered different doses of compound K (E-
2.1. Animals All animal procedures were performed to conform to NIH guidelines (Guide for the Care and Use of Laboratory Animals) and were performed in accordance with Guidelines for the Care and Use of Laboratory Animals stipulated by the Ethics Committee of Shengjing Hospital Affiliated with China Medical University (Shenyang, China). One hundred and four old male Kunming mice (16 months-old) were obtained from the Laboratory Animal Center of Shengjing Hospital of 3
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
(caption on next page)
4
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
Fig. 3. Expression of inflammatory cytokines and genes downstream of LXRα in hippocampal tissues. (C: control group; Model: model group; L: 3 mg/kg compound K group; M: 10 mg/kg compound K group; H: 30 mg/kg compound K group; V: Vehicle group; C + M: control + 10 mg/kg compound K group). A. Immunohistochemistry in mouse hippocampal tissues showed a significant decrease in the expression of IL-1β caused by the treatment of Compound K. B. TNF-α expression in mouse hippocampal tissues was reduced significantly by the administration of compound K. C. Immunoblotting analysis showed a significant decrease in the levels of IL-1β, TNF-α, and NF-κB P65 in mouse hippocampal tissues after the administration of compound K. D. Relative mRNA levels of ABCA1, ABCG1, and apoE in mouse hippocampal tissues were increased significantly after treatment with compound K. *p < 0.05. Scale bar = 100 μm. Data are presented as the mean ± SD. n = 8.
mouse was released from the contralateral quadrant of original platform quadrant. The frequency of crossing and the time the mouse spent in the original platform quadrant were recorded.
0120, Tauto Biotech Co., Ltd., Shanghai, China) (0, 3, 10, or 30 mg/kg, intra-peritoneally, quaque die) from day 8 to day 14 after partial hepatectomy. Another group of mice were treated with 10 mg/kg compound K after a sham operation. Reagent solutions were prepared as followed: the reagents were dissolved in 100% dimethyl sulfoxide (DMSO) as 100 X stock solutions respectively, compound K (30 mg/mL, 100 mg/mL or 300 mg/mL), GGPP (90 mg/mL), atorvastatin (26 mg/ mL) and GW3965 (30 mg/mL). 2 u L stock solution was diluted with 198 u L sterile PBS as 1 X injective solution for 20 g mouse. 1% DMSO in sterile PBS was used as the vehicle control. Animals were fasted overnight and euthanized at day 15 after operation; blood samples were collected from the abdominal vena cava. Animals were sacrificed by CO2 inhalation following the AVMA guidelines for the euthanasia of animals (2013 edition). The hippocampus was dissected for the sequential experiments. To explore the possible mechanism of compound K on POCD, fortyeight mice were divided into six groups (n = 8) randomly. Mice in the control group, model group, and compound K (10 mg/kg) group were treated as indicated above. Mice in the GGPP group were administered an LXR inhibitor, geranylgeranyl pyrophosphate ammonium salt (GGPP, G6025, Sigma-Aldrich, St. Louis, MO) (9 mg/kg, intra-peritoneally, quaque die) based on the dosages used in the compound K group. Animals in the atorvastatin and GW3965 groups were administered 2.6 mg/kg atorvastatin calcium (PHR1422, Sigma-Aldrich) or 3 mg/kg GW3965 hydrochloride (G6295, Sigma-Aldrich) (intra-peritoneally, quaque die) from day 8 to day 14 after partial hepatectomy, respectively.
2.4. Immunohistochemistry Hippocampal tissues were fixed by perfusion with 10% formalin and sectioned at 5 μm using a Leica RM2245 Cryostat (Leica Microsystems, Buffalo Grove, IL). After dewaxing and rehydration, slides were immersed in sub-boiling 10 mM sodium citrate (pH 6.0), and then treated with Dako enzyme block (Dako, Carpinteria, CA). Slides, blocked with 5% bovine serum albumin (BSA, A602449, Sangon Biotech, Shanghai, China) for 30 min, were washed in PBS with 0.1% Triton 100 (PBSTr) and overlaid with anti-tumor necrosis factor-α (TNF-α) antibody (Santa Cruz, sc-52746, 1:300) or anti-cleaved interleukin-1β (IL-1β) antibody (Santa Cruz, sc-23460, 1:300) for 2 h at ambient temperature, followed by species-specific horseradish peroxidase (HRP) -conjugated secondary antibodies (1:2000 in PBSTr) for 1 h. After washing three times with PBS containing 0.1% Tween 20 (PBSTw), binding was detected using an HRP-DAB Substrate Kit (PA110, Tiangen, Beijing, China). The positive staining (brown) of pro-inflammatory cytokines, IL-1β and TNF-α, was observed in hippocampus, especially in granule cell layer of the dentate gyrus and CA1 region. 2.5. Determination of serum lipid, glucose, and cytokine levels Each blood sample was centrifuged by 1000×g for 10 min at 4 °C; the supernatant was collected as the serum sample. The levels of serous triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were detected using an AU-2700 automatic biochemical analyzer (Olympus, Tokyo, Japan). Detection kits were obtained from Kang Taike Medical Co., Ltd. (Beijing, China) and the standard sample was purchased from Randox Laboratories Ltd. (Northern Ireland, UK). Serum glucose levels were detected using a Beckman AU5800 (Miami, FL, USA) glucose meter; the standard sample was purchased from Roche (Roche, Basel, Switzerland). The levels of serum TNF-α and IL-1β were detected with Mcytomag-70 K, a mouse cytokine/chemokine magnetic bead panel (Merck Millipore Co., Ltd., Billerica, MA, USA).
2.2. Partial hepatectomy The partial hepatectomy in mice was performed as we have previously reported [24]. Briefly, the animals were anesthetized with 10% chloral hydrate. On the upper abdomen, a midline incision was performed to expose the liver, then the left hepatic duct and artery were ligated tightly with No.10 silk. After ischemia had presented, the left lateral and median lobes of liver were removed carefully. Then, the peritoneum and skin were sutured with silk. During the operation, arterial blood O2 partial pressure and CO2 partial pressure were kept at 100–120 mmHg and 35–45 mmHg, respectively. Light-emitting diode shower lights were used to keep body temperature at 37 ± 0.5 °C.
2.6. Real-time PCR 2.3. Morris Water-Maze (MWM) test Total RNA from hippocampus tissue was extracted using an RNAsimple Total RNA Kit (DP419, Tiangen); cDNA was obtained using a PrimeScript™ RT reagent Kit with gDNA Eraser (RR047A, TaKaRa, Tokyo, Japan). Real-time PCR was performed using SYBR® Premix Ex Taq™ II (RR820A, TaKaRa) on a REALPLEX (Eppendorf, Hamburg, Germany) reaction system under the following conditions: 30 s at 95 °C, 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Primers were designed as follows: for mouse ABCA1 were 5′-ACATCCTCGTCCATTAAGCC-3′ and 5′-AACTCTGGCACACTCATTGC-3′; for mouse ABCG1 were 5′-TGTCA GCTTTGACACCATCC-3′ and 5′-GGTACAGCTTCGCATTCTCC-3′; for mouse ApoE were 5′-TTTATTAAGCAAGGGCCACC-3′ and 5′-CTTCTCC TGTCCTGCAACAA-3′; and for mouse β-actin were 5′-ATTGAACATGG CATTGTTACC-3′ and 5′-GGCATACAGGGACAGCACAGC-3′. The fold alteration relative to the control sample was determined using the 2ΔΔCt (cycle threshold) method; the Ct value was normalized to the β-
To evaluate spatial memory and learning abilities, mice underwent the MWM test [26] on day 10 and day 14 after partial hepatectomy. Briefly, for training the mice on day 8 and day 9, each mouse was released into the water from one of the four quadrants facing the wall of the test pool (The Morris Water Maze, XR-XM101, Shanghai XinRuan information Technology Co., Ltd., Shanghai, China); then, the time for mouse to reach the platform placed in the center of the pool was recorded. The mouse had to locate and land on the platform within 60 s, otherwise it was picked up and placed on the platform and the time was recorded as 60 s. To strengthen memory, the animal was allowed to stay on the platform for 15 s. For navigation experiment on day 10 and day 14, the test above was repeated with entry from each of the four quadrants and the escape latency time was recorded. On day 11 and 15, the probe test session was continued in the pool without a platform. The 5
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
Fig. 4. Compound K attenuation of POCD symptoms occurs via LXRα activation. A. Treatment with GGPP reversed the effects of compound K on Morris water-maze (MWM) test scores in post-hepatectomy mice. B. Serum cytokine levels were increased significantly as compared with the compound K group. C. GGPP reversed the effects of compound K on serum lipid levels in post-hepatectomy mice. D. Immunoblotting analysis showed the treatment of GGPP could increase the levels of inflammatory cytokines in hippocampal tissue as compared with the compound K group. E. The treatment of GGPP significantly decreased the levels of LXRα downstream genes in hippocampal tissues as compared with the compound K group. *p < 0.05. Data are presented as the mean ± SD. n = 8. 6
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
actin expression level. Experiments were performed in quintuplicate.
3.3. Compound K reduced cytokine levels in the hippocampus of aged mice following partial hepatectomy
2.7. Immunoblotting
TNF-α and IL-1β protein levels were measured in the hippocampus of aged mice via immunohistochemistry and immunoblotting at day 15 after surgery. Both IL-1β (Fig. 3A) and TNF-α (Fig. 3B) expression were significantly increased in the model group, while the treatment of compound K (10, 30 mg/kg) significantly attenuated the alteration of cytokine levels (Fig. 3A, B). Compound K inhibited the expression of inflammatory cytokines in the hippocampus that resulted from partial hepatectomy. Compared with model group, TNF-α, IL-1β, and NF-κB P65 levels were significantly decreased in the group receiving compound K (10, 30 mg/kg) (Fig. 3C). The immunoblotting results were consistent with those obtained via immunohistochemistry analysis on hippocampus. There was no statistical difference in the levels of inflammatory cytokines between the control group and the group receiving only compound K. The mRNA levels of genes downstream of LXR, including ABCA1, ABCG1, and ApoE, were significantly increased after treatment of compound K (10, 30 mg/kg) in both the surgical group as well as the group treated only with compound K (Fig. 3D). The expressions of TNF-α and IL-1β were correlated with MWM scores (Fig. S1).
Protein samples from freshly obtained hippocampus tissue was extracted using Tissue Protein Extraction Reagent (78510, Thermo Fisher Scientific, Waltham, MA, USA), and resolved by SDS-gel electrophoresis on 10% NuPAGE Bis-Tris polyacrylamide gels for 50 min at 150 V. Proteins were transferred onto PVDF membranes and blocked with 5% BSA in PBSTw for 30 min. Membranes were washed three times in PBSTw and incubated with primary antibodies for 16 h at 4 °C, independently. After three washes, blots were incubated with horseradish peroxidase (HRP) -conjugated secondary antibodies (1:5000 in PBSTw) for 1 h at ambient temperature. After three washes, antibody binding was visualized using enhanced chemiluminescence (ECL, GE Healthcare, Little Chalfont, Buckinghamshire, UK). As a loading control, samples were incubated with the anti-β-actin antibody (1:2000, Kangchen Inc., Shanghai, China) described above. Blot images were obtained using a Chemidoc-XRS image system (Bio-Rad, Hercules, CA, USA); bands were quantified with Quantity One software (Bio-Rad) and normalized to β-actin expression. The following primary antibodies were used: anti-TNF-α (1:500), anti-cleaved IL-1β (1:500), and anti-NFκB p65 (Santa Cruz, sc-398442, 1:500).
3.4. The attenuation of POCD by compound K occur via LXR activation Compared with the model group, treatment with 10 mg/kg compound K, but not atorvastatin, attenuated symptoms of cognitive dysfunction after partial hepatectomy in aged mice (Fig. 4A). Treatment with atorvastatin calcium decreased both the levels of serum TNF-α (Fig. 4B) and LDL (Fig. 4C). Treatment with compound K decreased levels of serum TNF-α, IL-1β (Fig. 4B), and LDL (Fig. 4C) and also increased levels of HDL (Fig. 4C). Compared with the model group, treatment with 10 mg/kg compound K, but not atorvastatin, decreased levels of TNF-α and IL-1β in the hippocampus (Fig. 4D). There were significant differences in mRNA levels of ABCA1, ABCG1, and ApoE in hippocampal tissues between the compound K only group and the model group (Fig. 4E). Treatment with GGPP reversed all of effects induced by compound K.
2.8. Statistical analysis All data were expressed as the mean ± SD. Histograms methods was used to evaluate the assumptions of the data. Comparisons between groups were conducted using one-way analysis of variance (ANOVA) plus a post-hoc least significant difference (LSD) test. All analyses were performed using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA). Values of p < 0.05 were considered to be statistically significant.
3. Results 3.1. Compound K attenuated transient POCD caused by partial hepatectomy in aged mice
4. Discussion
The MWM tests were used to determine cognitive impairment induced by partial hepatectomy. Mice in the model group exhibited prolonged escape latency, shortened original quadrant time, and decreased frequency of crossing at both days 10 and 14 as compared with the control group (p < 0.05). These results indicate that spatial memory was significantly impaired after the partial hepatectomy. Treatment with compound K (10, 30 mg/kg) improved symptoms of cognitive dysfunction on all of the MWM tests conducted, when compared with mice in the model group (Fig. 1). The pharmacological effect of compound K was shown to be dose-dependent. Compared with control group, administration of 10 mg/kg compound K did not affect the spatial memory significantly.
During recent years, POCD has become a growing concern for surgical doctors and their support teams. Dysfunctions in short-term and long-term memory, mood, and consciousness can be detected days to weeks after surgical operations and can persist for weeks to months [27]. Aging is well known as a major risk factor of POCD. Compared with young and middle-aged people, a higher ratio of elderly patients suffers from POCD; cognitive dysfunction lasts three months in 14% of patients over sixty years old and symptoms are independent of both the type of operation and anesthetic [28,29]. The pathological mechanism of POCD is not yet completely understood, therefore the therapies are limited. In addition to exclusion of any other disease associated with organic psychosyndrome, and based on the symptoms, the present treatments of POCD are limited to providing sufficient ventilation and oxygenation, hemodynamic support, pain control, promotion of normal circadian rhythms, and maintenance of electrolyte balance [30,31]. Studies have indicated important roles of systemic and local inflammatory cytokines in the presentation of POCD. Our previous study reported hydrogen-rich saline could attenuate the symptoms of POCD via inflammation inhibition [25] and the treatment with thalidomide could attenuate the spatial memory deficit in aged rats after laparotomy by reducing TNF-α and IL-1β levels [32]. These results indicated that anti-inflammatory treatment might be a potential effective method to attenuate POCD. Based on our previous study [10] on the LXRα activation of compound K (310 mg/kg) in mice, and Seo’s [33] and Hou’s [34] studies on the neuroprotective, hippocampal neurogenesis and
3.2. Compound K modulated systemic inflammatory cytokines and serum lipids of aged mice following partial hepatectomy There was no significant difference in serum glucose levels (Fig. 2A) or body weight (Fig. 2B) among the groups. Serum TNF-α and IL-1β levels were significantly increased after partial hepatectomy. Treatment with compound K resulted in decreased levels of cytokines in a dosedependent manner (Fig. 2C). Compared with mice in the model group, serum LDL-C levels were decreased in mice after the administration of compound K (10, 30 mg/kg), while HDL-C levels were significantly increased (Fig. 2D). 7
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
Author contributions
cognition-enhancing effects of compound K (5, 10 mg/kg) in mice, we explored the effects of compound K from 3 mg/kg to 30 mg/kg on POCD in mice. The present study showed that compound K could significantly attenuate the memory dysfunction of aged mice after partial hepatectomy in all MWM tests in a dose-dependent manner; our previous study indicated that compound K was a novel agonist of LXRα [35]. Therefore, we hypothesized that the role of compound K might be influence activation of LXRα. Here, we report that treatment with GGPP reverses the effects of compound K, thus confirming that the pharmacological effects of compound K are dependent on LXRα activation. LXRα is a regulator of reverse cholesterol transport. Activation of LXRα up-regulates expression of the downstream genes such as ABCA1, ABCG1, and ApoE and induces multiple biological alterations including the regulation of serum lipids and inhibition of inflammation [36]. To explore whether serum lipid alteration is involved in the mechanism by which compound K influences POCD, we used atorvastatin calcium as a control. Compared with the model group, treatment with atorvastatin calcium resulted in decreased levels of LDL and TC, as well as serum TNF-α. However, there was no significant difference in MWM tests results between mice in the atorvastatin calcium group and the model group. These results indicate that serum lipid levels do not play an important role in POCD symptoms. Therefore, we can conclude that the effect of serum lipid modulation by compound K did not contribute to the attenuation of POCD. In addition, our results show that systemic cytokines levels were also independent of POCD presentation. This result is consistent with clinical research, indicating that systemic noninfective inflammation is not associated with the development of POCD during the early post-operative period in elderly patients [37]. In the immunohistochemistry analysis, levels of TNF-α and IL-1β in hippocampus tissue exhibited a direct relationship to MWM scores. Treatment with compound K could decrease levels of TNF-α and IL-1β in hippocampal tissue in a dose-dependent manner. A previous study indicated that LXR agonists could alleviate brain inflammation by acting on upregulation of ABCA1 expression in neurons, astrocytes, and microglia [21], which is consistent with our results. Therefore, all these results indicate that the anti-inflammatory effect in local hippocampus tissue plays a key role in the elucidation of the mechanism by which compound K on influences POCD. Increased levels genes downstream of LXRα after hepatectomy indicate that activation of the LXRα pathway might be a protective reaction, which was not strong enough to completely attenuate the inflammation. Compound K, as an agonist, could activate the anti-inflammatory pathway, thereby attenuating the symptoms of POCD. Our previous study demonstrated that compound K was a novel agonist of LXRα without serious side effects [10,35] such as lipid deposition in liver, which are induced by general LXRα activators currently in use including GW3965 and T0901317. Furthermore, other studies have shown that compound K can attenuate fatty liver in rats [38]. The safety of compound K has been confirmed by several reports [39,40]. Therefore, the present study demonstrates a potential therapeutic role for compound K in POCD.
Y. Tian conceived and designed the experiments; Q. Liu and L. Liu performed the in vivo experiments, including operation and samples collection; H. Liu performed the cell experiments; C. Wang contributed Real-time PCR; J. Jiang and Y. Jia analyzed the data; S. Guo contributed MWM tests; Y. Tian, Y. Jia and Q. Liu wrote the paper. All authors have revised the manuscript and given final approval to the version to be published. Declaration of Competing Interest The authors have declared that no conflict of interest exists. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109400. References [1] A. Rudra, S. Chatterjee, J. Kirtania, S. Sengupta, G. Moitra, S. Sirohia, et al., Postoperative delirium, Indian J. Crit. Care Med. 10 (2006) 235–240, https://doi. org/10.4103/0972-5229.29842. [2] C.C. Price, C.W. Garvan, T.G. Monk, Type and severity of cognitive decline in older adults after noncardiac surgery, Anesthesiology 108 (2008) 8–17, https://doi.org/ 10.1097/01.anes.0000296072.02527.18. [3] K.R. Murrin, T.M. Nagarajan, Hyperventilation and psychometric testing. A preliminary study, Anaesthesia 29 (1974) 50–58, https://doi.org/10.1111/j.13652044.1974.tb00582.x. [4] J.T. Moller, P. Cluitmans, L.S. Rasmussen, P. Houx, H. Rasmussen, J. Canet, et al., Long-term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International study of post-operative cognitive dysfunction, Lancet. 351 (1998) 857–861, https://doi.org/10.1016/S0140-6736(97)07382-0. [5] P.A. Barber, S. Hach, L.J. Tippett, L. Ross, A.F. Merry, P. Milsom, Cerebral ischemic lesions on diffusion weighted imaging are associated with neurocognitive decline after cardiac surgery, Stroke 39 (2008) 1427–1433, https://doi.org/10.1161/ STROKEAHA.107.50298. [6] B. Jungwirth, K. Kellermann, M. Qing, G.B. Mackensen, M. Blobner, E.F. Kochs, Cerebral tumor necrosis factor α expression and long-term neurocognitive performance after cardiopulmonary bypass in rats, J. Thorac. Cardiovasc. Surg. 138 (2009) 1002–1007, https://doi.org/10.1016/j.jtcvs.2009.06.022. [7] V. Tomic, S. Russwurm, E. Möller, R.A. Claus, M. Blaess, F. Brunkhorst, et al., Transcriptomic and proteomic patterns of systemic inflammation in on-pump and off-pump coronary artery bypass grafting, Circulation. 112 (2005) 2912–2920, https://doi.org/10.1161/CIRCULATIONAHA.104.531152. [8] Y. Ding, C. Shi, L. Chen, P. Ma, K. Li, J. Jin, et al., Effects of andrographolide on postoperative cognitive dysfunction and the association with NF-κB/MAPK pathway, Oncol. Lett. 14 (2017) 7367–7373, https://doi.org/10.3892/ol.2017. 7088. [9] J.A. Clemens, D.T. Stephenson, E.B. Smalstig, E.P. Dixon, S.P. Little, Global ischemia activates nuclear factorb in forebrain neurons of rats, Stroke. 28 (1997) 1073–1080, https://doi.org/10.1161/01.STR.28.5.1073. [10] L. Zhou, Y. Zheng, Z. Li, L. Bao, Y. Dou, Y. Tang, et al., Compound K attenuates the development of atherosclerosis in ApoE-/- mice via LXRα activation, Int. J. Mol. Sci. 17 (2016) 1054, https://doi.org/10.3390/ijms17071054. [11] S. Kang, M.H. Siddiqi, S.J. Yoon, S. Ahn, H.Y. Noh, N.S. Kumar, et al., Therapeutic potential of compound K as an IKK inhibitor with implications for osteoarthritis prevention: an in silico and in vitro study, In Vitro Cell. Dev. Biol. Anim. 52 (2016) 895–905, https://doi.org/10.1007/s11626-016-0062-9. [12] R. Wang, M. Zhang, S. Hu, K. Liu, Y. Tai, J. Tao, et al., Ginsenoside metabolite compound-K regulates macrophage function through inhibition of β-arrestin2, Biomed. Pharmacother. 115 (2019) 108909, , https://doi.org/10.1016/j.biopha. 2019.108909. [13] S. Lee, M.C. Kwon, J.P. Jang, J.K. Sohng, H.J. Jung, The ginsenoside metabolite compound K inhibits growth, migration and stemness of glioblastoma cells, Int. J. Oncol. 51 (2017) 414–424, https://doi.org/10.3892/ijo.2017.405. [14] C. Hu, G. Song, B. Zhang, Z. Liu, R. Chen, H. Zhang, et al., Intestinal metabolite compound K of panaxoside inhibits the growth of gastric carcinoma by augmenting apoptosis via Bid-mediated mitochondrial pathway, J. Cell. Mol. Med. 16 (2012) 96–106, https://doi.org/10.1111/j.1582-4934.2011.01278.x. [15] Y.C. Hwang, D.H. Oh, M.C. Choi, S.Y. Lee, K.J. Ahn, H.Y. Chung, et al., Compound K attenuates glucose intolerance and hepatic steatosis through AMPK-dependent pathways in type 2 diabetic OLETF rats, Korean J. Intern. Med. (2017), https://doi. org/10.3904/kjim.2015.208. [16] W. Chen, J. Wang, Y. Luo, T. Wang, X. Li, A. Li, et al., Ginsenoside Rb1 and compound K improve insulin signaling and inhibit ER stress-associated NLRP3 inflammasome activation in adipose tissue, J. Ginseng Res. 40 (2016) 351–358. [17] E. Kim, D. Kim, S. Yoo, Y.H. Hong, S.Y. Han, S. Jeong, et al., The skin protective
5. Conclusions Overall, the present work sheds light on the alleviation of POCD by compound K via inhibition of the local inflammatory response in hippocampal tissue; furthermore, this mechanism is likely to be associated with LXRα activation. These findings suggest further evaluation of compound K as a potential effective modulator for POCD is needed.
Funding This work was supported by the National Natural Science Foundation of China (No. 81701075). 8
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
19434.1e. [29] L. Evered, D.A. Scott, B. Silbert, P. Maruff, Postoperative cognitive dysfunction is independent of type of surgery and anesthetic, Anesth. Analg. 112 (2011) 1179–1185, https://doi.org/10.1213/ANE.0b013e318215217e. [30] W. Wang, Y. Wang, H. Wu, L. Lei, S. Xu, X. Shen, et al., Postoperative cognitive dysfunction: current developments in mechanism and prevention, Med. Sci. Monit. 20 (2014) 1908–1912, https://doi.org/10.12659/MSM.892485. [31] M. Pappa, N. Theodosiadis, A. Tsounis, P. Sarafis, Pathogenesis and treatment of post-operative cognitive dysfunction, Electron. Physician 9 (2017) 3768–3775, https://doi.org/10.19082/3768. [32] P. Guo, S.P. Hu, Thalidomide alleviates postoperative pain and spatial memory deficit in aged rats, Biomed. Pharmacother. 95 (2017) 583–588, https://doi.org/10. 1016/j.biopha.2017.08.114. [33] J.Y. Seo, S.H. Ju, J. Oh, S.K. Lee, J.S. Kim, Neuroprotective and cognition-enhancing effects of compound K isolated from red ginseng, J. Agric. Food Chem. 64 (2016) 2855–2864, https://doi.org/10.1021/acs.jafc.5b05789. [34] J.G. Hou, J.J. Xue, M.R. Lee, M.Q. Sun, X.H. Zhao, Y.N. Zheng, et al., Compound K is able to ameliorate the impaired cognitive function and hippocampal neurogenesis following chemotherapy treatment, Biochem. Biophys. Res. Commun. 436 (2013) 104–109, https://doi.org/10.1016/j.bbrc.2013.05.087. [35] Y. Huang, H. Liu, Y. Zhang, J. Li, C. Wang, L. Zhou, et al., Synthesis and biological evaluation of ginsenoside compound K derivatives as a novel class of LXRα activator, Molecules. 22 (2017) 1232, https://doi.org/10.3390/molecules22071232. [36] S.S. Im, T.F. Osborne, Liver X receptors in atherosclerosis and inflammation, Circ. Res. 108 (2011) 996–1001, https://doi.org/10.1161/CIRCRESAHA.110.226878. [37] E. Nemeth, K. Vig, K. Racz, K.B. Koritsanszky, K.I. Ronkay, F.P. Hamvas, et al., Influence of the postoperative inflammatory response on cognitive decline in elderly patients undergoing on-pump cardiac surgery: a controlled, prospective observational study, BMC Anesthesiol. 17 (2017) 113, https://doi.org/10.1186/ s12871-017-0408-1. [38] X.J. Chen, W.J. Liu, M.L. Wen, H. Liang, S.M. Wu, Y.Z. Zhu, et al., Ameliorative effects of Compound K and ginsenoside Rh1 on non-alcoholic fatty liver disease in rats, Sci. Rep. 7 (2017) 41144, https://doi.org/10.1038/srep41144. [39] Y.L. Gao, Z.F. Liu, C.M. Li, J.Y. Shen, H.X. Yin, G.S. Li, Subchronic toxicity studies with ginsenoside compound K delivered to dogs via intravenous administration, Food Chem. Toxicol. 49 (2011) 1857–1862, https://doi.org/10.1016/j.fct.2011. 05.00. [40] L. Chen, L. Zhou, J. Huang, Y. Wang, G. Yang, Z. Tan, et al., Single- and multipledose trials to determine the pharmacokinetics, safety, tolerability, and sex effect of oral ginsenoside compound k in healthy Chinese volunteers, Front. Pharmacol. 8 (2018) 965, https://doi.org/10.3389/fphar.2017.00965.
effects of compound K, a metabolite of ginsenoside Rb1 from Panax ginseng, J. Ginseng Res. 42 (2018) 218–224. J. Oh, J.S. Kim, Compound K derived from ginseng: neuroprotection and cognitive improvement, Food Funct. 7 (2016) 4506–4515, https://doi.org/10.1039/ c6fo01077f. J. Chen, H. Wu, Q. Wang, Y. Chang, K. Liu, W. Wei, Ginsenoside metabolite Compound K suppresses T-cell priming via modulation of dendritic cell trafficking and costimulatory signals, resulting in alleviation of collagen-induced arthritis, J. Pharmacol. Exp. Ther. 353 (2015) 71–79, https://doi.org/10.1124/jpet.114. 220665. J. Chen, M. Si, Y. Wang, L. Liu, Y. Zhang, A. Zhou, et al., Ginsenoside metabolite compound K exerts anti-inflammatory and analgesic effects via downregulating COX2, Inflammopharmacology. 27 (2019) 157–166, https://doi.org/10.1007/ s10787-018-0504-y. I. Lefterov, A. Bookout, Z. Wang, M. Staufenbiel, D. Mangelsdorf, R. Koldamova, Expression profiling in APP23 mouse brain: inhibition of Abeta amyloidosis and inflammation in response to LXR agonist treatment, Mol. Neurodegener. 2 (2007) 20, https://doi.org/10.1186/1750-1326-2-20. A. Archer, E. Stolarczyk, M.L. Doria, L. Helguero, R. Domingues, J.K. Howard, et al., LXR activation by GW3965 alters fat tissue distribution and adipose tissue inflammation in ob/ob female mice, J. Lipid Res. 54 (2013) 1300–1311, https://doi. org/10.1194/jlr.M033977. E. Vucic, C. Calcagno, S.D. Dickson, J.H. Rudd, K. Hayashi, J. Bucerius, et al., Regression of inflammation in atherosclerosis by the LXR agonist R211945: a noninvasive assessment and comparison with atorvastatin, JACC Cardiovasc. Imaging 5 (2012) 819–828, https://doi.org/10.1016/j.jcmg.2011.11.025. C. Hu, A.J. Lau, R. Wang, T.K.H. Chang, Comparative analysis of ginsenosides in human glucocorticoid receptor binding, transactivation, and transrepression, Eur. J. Pharmacol. 815 (2017) 501–511, https://doi.org/10.1016/j.ejphar.2017.10.019. Y. Tian, S. Guo, Y. Zhang, Y. Xu, P. Zhao, X. Zhao, Effects of hydrogen-rich saline on hepatectomy-induced postoperative cognitive dysfunction in old mice, Mol. Neurobiol. 54 (2017) 2579–2584, https://doi.org/10.1007/s12035-016-9825-2. Y. Tian, S. Guo, L. Ma, Y. Wang, Y. Xu, P. Zhao, Sevoflurane aggregates cognitive dysfunction and hippocampal oxidative stress induced by β-amyloid in rats Author links open overlay panel, Life Sci. 143 (2015) 194–201, https://doi.org/10.1016/j. lfs.2015.11.002. L. Krenk, L.S. Rasmussen, H. Kehlet, New insights into the pathophysiology of postoperative cognitive dysfunction, Acta Anaesthesiol. Scand. 54 (2010) 951–956, https://doi.org/10.1111/j.1399-6576.2010.02268. T.G. Monk, B.C. Weldon, C.W. Garvan, D.E. Dede, M.T. van der Aa, K.M. Heilman, et al., Predictors of cognitive dysfunction after major noncardiac surgery, Anesthesiology. 108 (2008) 18–30, https://doi.org/10.1097/01.anes.0000296071.
9
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Compound K attenuated hepatectomy-induced post-operative cognitive dysfunction in aged mice via LXRα activation
T
Qifang Liua, Lidan Liub, Hongmei Liuc, Jingjing Jiangb, Shanbin Guod, Cong Wangb, Yi Jiac, ⁎ Yue Tianb, a
Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang, 110004, China Department of Anesthesiology, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang, 110004, China c Institute of Materia Medica and Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, 30 Gaotanyan Street, Chongqing, 400038, China d Department of Pharmacy, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang, 110004, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Compound K Inflammation Liver X receptor Post-operative cognitive dysfunction
Aims: Post-operative cognitive dysfunction (POCD) occurs after major surgery in elderly patients and affects the quality of patients’ lives. The present study aims to explore the protective effects and possible mechanisms of compound K in old mice with POCD caused by partial hepatectomy. Methods: Sixteen month-old mice were administered different doses of compound K from the 8th day to 14th day after partial hepatectomy. Cognitive function was subsequently measured with a Morris water-maze (MWM) test. Serum inflammatory cytokine levels were measured by magnetic bead panel; levels of cytokines in the hippocampus were analyzed using immunohistochemistry and immunoblotting. The mRNA levels of target genes were measured using real-time PCR. Results: Compared with the model group, MWM scores were significantly attenuated at days 10 and 14 postsurgery in mice receiving compound K (10, 30 mg/kg) in a dose-dependent manner. Both systemic and local cytokine levels were reduced after treatment of compound K. The alterations in serum lipids were independent of the attenuation of POCD syndrome. An inhibitor of liver X receptor-α (LXRα), GGPP, reversed the effects of compound K. Conclusions: The results provide evidence for an alleviation of POCD by compound K via local inflammation inhibition in hippocampus tissue; furthermore, the data suggests the mechanism involves the LXRα pathway. The present study supports further evaluation of compound K as a potential effective modulator for POCD.
1. Introduction Post-operative cognitive dysfunction (POCD), which is defined by various alterations of a patient’s cognitive functioning compared to their pre-surgery status, is one of the cognitive disorders found in the postoperative period [1]. POCD occurs frequently after patients undergo certain major surgical operations, especially in the elderly, and seriously affects patients’ quality of life [2]. The pathophysiology and possible mechanism of POCD is less well defined. Hyperventilation [3], hypotension [4], cerebral microemboli [5], and inflammation [6] are thought to be involved in the process of POCD. As indicated in many previous studies, systemic and cerebral inflammatory reactions [7], especially the activation of the NF-κB signaling pathway [8,9], play a major role in POCD. Hence the regulation of inflammation might be a potential effective therapeutic target for POCD, in addition to other
⁎
symptomatic treatments. Panax notoginseng (Burk.) F. H. Chen has been widely used as a major natural remedy in traditional medicine for the recovery of trauma for about 1000 years in Asia. Our previous studies have showed that one of the dammarane-type triterpene active metabolic components of Panax notoginseng saponins, 20-O-d-glucopyranosyl-20(S)-protopanaxadiol (compound K, 10 and 30 mg/kg in mice), can reduce inflammatory reactions in atherosclerosis lesions via liver X receptor α (LXRα) activation [10]. In addition, multiple other pharmaceutical activities have been reported for compound K, including anti-arthritis [11,12], anti-tumor [13,14], anti-diabetes [15,16], skin protection [17], neuroprotection [18] and anti-inflammation [19,20]. Recent studies have clearly shown the importance of the LXR pathway in both the inflammatory reaction and general immunological regulation. For example, GW3965, an LXR agonist, has been shown to alleviate the
Corresponding author. E-mail address: [email protected] (Y. Tian).
https://doi.org/10.1016/j.biopha.2019.109400 Received 13 June 2019; Received in revised form 22 August 2019; Accepted 28 August 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
Fig. 1. Compound K (CK) improved Morris water-maze (MWM) test scores of POCD mice. A. The administration of compound K (10, 30 mg/kg) reduced the escape latency (time until reaching the platform, day 10 and 14) as compared to the model group. B. The administration of compound K (10, 30 mg/kg) increased the time spent in the original platform quadrant as compared with the model group (day 11 and 15). C. Treatment with compound K (10, 30 mg/kg) increased the total number of crossings of the original platform quadrant of mice as compared with the model group (day 11 and 15). *p < 0.05. Data are presented as the mean ± SD. n = 8.
compound K acts on POCD. Towards this end, we evaluated compound K using an animal model of POCD induced by partial hepatectomy in old mice, as we have previously reported [25].
inflammation reaction in brain [21], adipose tissue [22] and atherosclerosis lesions [23], independent with the activation of glucocorticoid receptor [24]. Therefore, the present study was undertaken to further understand the protective effect and underlying mechanisms by which 2
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
Fig. 2. Effects of compound K (CK) on serum glucose, weight, serum inflammatory cytokines, and lipids levels in aged mice post-surgery. A. There was no significant difference of serum glucose levels among the groups. B. There was no significant difference of body weights among the groups. C. Serum inflammatory cytokines (TNF-α and IL-1β) in the model group were increased significantly as compared with the control group; furthermore, treatment with compound K (10, 30 mg/kg) significantly decreased levels of inflammatory cytokines as compared with the model group. D. There was no significant difference in serum triglyceride or total cholesterol levels among the groups. The administration of compound K (10, 30 mg/kg) significantly decreased LDL levels and increased HDL levels as compared with the model group. *p < 0.05. Data are presented as the mean ± SD. n=8.
2. Methods
China Medical University. All animals underwent a common one-week acclimatization period and were housed singly in a temperature-controlled (22–25 °C) environment on a 12 h light-dark cycle, under specific pathogen-free conditions, with food and water given ad libitum. The body weight of each animal was monitored every three days. To explore the potential effects of compound K on POCD, the mice were randomly divided into seven groups (n = 8). The mice in model group underwent partial hepatectomy under anesthesia, while the animals in control group were underwent a sham operation (same protocol as model group, expect liver excision). Mice in vehicle and compound K groups were administered different doses of compound K (E-
2.1. Animals All animal procedures were performed to conform to NIH guidelines (Guide for the Care and Use of Laboratory Animals) and were performed in accordance with Guidelines for the Care and Use of Laboratory Animals stipulated by the Ethics Committee of Shengjing Hospital Affiliated with China Medical University (Shenyang, China). One hundred and four old male Kunming mice (16 months-old) were obtained from the Laboratory Animal Center of Shengjing Hospital of 3
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
(caption on next page)
4
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
Fig. 3. Expression of inflammatory cytokines and genes downstream of LXRα in hippocampal tissues. (C: control group; Model: model group; L: 3 mg/kg compound K group; M: 10 mg/kg compound K group; H: 30 mg/kg compound K group; V: Vehicle group; C + M: control + 10 mg/kg compound K group). A. Immunohistochemistry in mouse hippocampal tissues showed a significant decrease in the expression of IL-1β caused by the treatment of Compound K. B. TNF-α expression in mouse hippocampal tissues was reduced significantly by the administration of compound K. C. Immunoblotting analysis showed a significant decrease in the levels of IL-1β, TNF-α, and NF-κB P65 in mouse hippocampal tissues after the administration of compound K. D. Relative mRNA levels of ABCA1, ABCG1, and apoE in mouse hippocampal tissues were increased significantly after treatment with compound K. *p < 0.05. Scale bar = 100 μm. Data are presented as the mean ± SD. n = 8.
mouse was released from the contralateral quadrant of original platform quadrant. The frequency of crossing and the time the mouse spent in the original platform quadrant were recorded.
0120, Tauto Biotech Co., Ltd., Shanghai, China) (0, 3, 10, or 30 mg/kg, intra-peritoneally, quaque die) from day 8 to day 14 after partial hepatectomy. Another group of mice were treated with 10 mg/kg compound K after a sham operation. Reagent solutions were prepared as followed: the reagents were dissolved in 100% dimethyl sulfoxide (DMSO) as 100 X stock solutions respectively, compound K (30 mg/mL, 100 mg/mL or 300 mg/mL), GGPP (90 mg/mL), atorvastatin (26 mg/ mL) and GW3965 (30 mg/mL). 2 u L stock solution was diluted with 198 u L sterile PBS as 1 X injective solution for 20 g mouse. 1% DMSO in sterile PBS was used as the vehicle control. Animals were fasted overnight and euthanized at day 15 after operation; blood samples were collected from the abdominal vena cava. Animals were sacrificed by CO2 inhalation following the AVMA guidelines for the euthanasia of animals (2013 edition). The hippocampus was dissected for the sequential experiments. To explore the possible mechanism of compound K on POCD, fortyeight mice were divided into six groups (n = 8) randomly. Mice in the control group, model group, and compound K (10 mg/kg) group were treated as indicated above. Mice in the GGPP group were administered an LXR inhibitor, geranylgeranyl pyrophosphate ammonium salt (GGPP, G6025, Sigma-Aldrich, St. Louis, MO) (9 mg/kg, intra-peritoneally, quaque die) based on the dosages used in the compound K group. Animals in the atorvastatin and GW3965 groups were administered 2.6 mg/kg atorvastatin calcium (PHR1422, Sigma-Aldrich) or 3 mg/kg GW3965 hydrochloride (G6295, Sigma-Aldrich) (intra-peritoneally, quaque die) from day 8 to day 14 after partial hepatectomy, respectively.
2.4. Immunohistochemistry Hippocampal tissues were fixed by perfusion with 10% formalin and sectioned at 5 μm using a Leica RM2245 Cryostat (Leica Microsystems, Buffalo Grove, IL). After dewaxing and rehydration, slides were immersed in sub-boiling 10 mM sodium citrate (pH 6.0), and then treated with Dako enzyme block (Dako, Carpinteria, CA). Slides, blocked with 5% bovine serum albumin (BSA, A602449, Sangon Biotech, Shanghai, China) for 30 min, were washed in PBS with 0.1% Triton 100 (PBSTr) and overlaid with anti-tumor necrosis factor-α (TNF-α) antibody (Santa Cruz, sc-52746, 1:300) or anti-cleaved interleukin-1β (IL-1β) antibody (Santa Cruz, sc-23460, 1:300) for 2 h at ambient temperature, followed by species-specific horseradish peroxidase (HRP) -conjugated secondary antibodies (1:2000 in PBSTr) for 1 h. After washing three times with PBS containing 0.1% Tween 20 (PBSTw), binding was detected using an HRP-DAB Substrate Kit (PA110, Tiangen, Beijing, China). The positive staining (brown) of pro-inflammatory cytokines, IL-1β and TNF-α, was observed in hippocampus, especially in granule cell layer of the dentate gyrus and CA1 region. 2.5. Determination of serum lipid, glucose, and cytokine levels Each blood sample was centrifuged by 1000×g for 10 min at 4 °C; the supernatant was collected as the serum sample. The levels of serous triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were detected using an AU-2700 automatic biochemical analyzer (Olympus, Tokyo, Japan). Detection kits were obtained from Kang Taike Medical Co., Ltd. (Beijing, China) and the standard sample was purchased from Randox Laboratories Ltd. (Northern Ireland, UK). Serum glucose levels were detected using a Beckman AU5800 (Miami, FL, USA) glucose meter; the standard sample was purchased from Roche (Roche, Basel, Switzerland). The levels of serum TNF-α and IL-1β were detected with Mcytomag-70 K, a mouse cytokine/chemokine magnetic bead panel (Merck Millipore Co., Ltd., Billerica, MA, USA).
2.2. Partial hepatectomy The partial hepatectomy in mice was performed as we have previously reported [24]. Briefly, the animals were anesthetized with 10% chloral hydrate. On the upper abdomen, a midline incision was performed to expose the liver, then the left hepatic duct and artery were ligated tightly with No.10 silk. After ischemia had presented, the left lateral and median lobes of liver were removed carefully. Then, the peritoneum and skin were sutured with silk. During the operation, arterial blood O2 partial pressure and CO2 partial pressure were kept at 100–120 mmHg and 35–45 mmHg, respectively. Light-emitting diode shower lights were used to keep body temperature at 37 ± 0.5 °C.
2.6. Real-time PCR 2.3. Morris Water-Maze (MWM) test Total RNA from hippocampus tissue was extracted using an RNAsimple Total RNA Kit (DP419, Tiangen); cDNA was obtained using a PrimeScript™ RT reagent Kit with gDNA Eraser (RR047A, TaKaRa, Tokyo, Japan). Real-time PCR was performed using SYBR® Premix Ex Taq™ II (RR820A, TaKaRa) on a REALPLEX (Eppendorf, Hamburg, Germany) reaction system under the following conditions: 30 s at 95 °C, 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Primers were designed as follows: for mouse ABCA1 were 5′-ACATCCTCGTCCATTAAGCC-3′ and 5′-AACTCTGGCACACTCATTGC-3′; for mouse ABCG1 were 5′-TGTCA GCTTTGACACCATCC-3′ and 5′-GGTACAGCTTCGCATTCTCC-3′; for mouse ApoE were 5′-TTTATTAAGCAAGGGCCACC-3′ and 5′-CTTCTCC TGTCCTGCAACAA-3′; and for mouse β-actin were 5′-ATTGAACATGG CATTGTTACC-3′ and 5′-GGCATACAGGGACAGCACAGC-3′. The fold alteration relative to the control sample was determined using the 2ΔΔCt (cycle threshold) method; the Ct value was normalized to the β-
To evaluate spatial memory and learning abilities, mice underwent the MWM test [26] on day 10 and day 14 after partial hepatectomy. Briefly, for training the mice on day 8 and day 9, each mouse was released into the water from one of the four quadrants facing the wall of the test pool (The Morris Water Maze, XR-XM101, Shanghai XinRuan information Technology Co., Ltd., Shanghai, China); then, the time for mouse to reach the platform placed in the center of the pool was recorded. The mouse had to locate and land on the platform within 60 s, otherwise it was picked up and placed on the platform and the time was recorded as 60 s. To strengthen memory, the animal was allowed to stay on the platform for 15 s. For navigation experiment on day 10 and day 14, the test above was repeated with entry from each of the four quadrants and the escape latency time was recorded. On day 11 and 15, the probe test session was continued in the pool without a platform. The 5
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
Fig. 4. Compound K attenuation of POCD symptoms occurs via LXRα activation. A. Treatment with GGPP reversed the effects of compound K on Morris water-maze (MWM) test scores in post-hepatectomy mice. B. Serum cytokine levels were increased significantly as compared with the compound K group. C. GGPP reversed the effects of compound K on serum lipid levels in post-hepatectomy mice. D. Immunoblotting analysis showed the treatment of GGPP could increase the levels of inflammatory cytokines in hippocampal tissue as compared with the compound K group. E. The treatment of GGPP significantly decreased the levels of LXRα downstream genes in hippocampal tissues as compared with the compound K group. *p < 0.05. Data are presented as the mean ± SD. n = 8. 6
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
actin expression level. Experiments were performed in quintuplicate.
3.3. Compound K reduced cytokine levels in the hippocampus of aged mice following partial hepatectomy
2.7. Immunoblotting
TNF-α and IL-1β protein levels were measured in the hippocampus of aged mice via immunohistochemistry and immunoblotting at day 15 after surgery. Both IL-1β (Fig. 3A) and TNF-α (Fig. 3B) expression were significantly increased in the model group, while the treatment of compound K (10, 30 mg/kg) significantly attenuated the alteration of cytokine levels (Fig. 3A, B). Compound K inhibited the expression of inflammatory cytokines in the hippocampus that resulted from partial hepatectomy. Compared with model group, TNF-α, IL-1β, and NF-κB P65 levels were significantly decreased in the group receiving compound K (10, 30 mg/kg) (Fig. 3C). The immunoblotting results were consistent with those obtained via immunohistochemistry analysis on hippocampus. There was no statistical difference in the levels of inflammatory cytokines between the control group and the group receiving only compound K. The mRNA levels of genes downstream of LXR, including ABCA1, ABCG1, and ApoE, were significantly increased after treatment of compound K (10, 30 mg/kg) in both the surgical group as well as the group treated only with compound K (Fig. 3D). The expressions of TNF-α and IL-1β were correlated with MWM scores (Fig. S1).
Protein samples from freshly obtained hippocampus tissue was extracted using Tissue Protein Extraction Reagent (78510, Thermo Fisher Scientific, Waltham, MA, USA), and resolved by SDS-gel electrophoresis on 10% NuPAGE Bis-Tris polyacrylamide gels for 50 min at 150 V. Proteins were transferred onto PVDF membranes and blocked with 5% BSA in PBSTw for 30 min. Membranes were washed three times in PBSTw and incubated with primary antibodies for 16 h at 4 °C, independently. After three washes, blots were incubated with horseradish peroxidase (HRP) -conjugated secondary antibodies (1:5000 in PBSTw) for 1 h at ambient temperature. After three washes, antibody binding was visualized using enhanced chemiluminescence (ECL, GE Healthcare, Little Chalfont, Buckinghamshire, UK). As a loading control, samples were incubated with the anti-β-actin antibody (1:2000, Kangchen Inc., Shanghai, China) described above. Blot images were obtained using a Chemidoc-XRS image system (Bio-Rad, Hercules, CA, USA); bands were quantified with Quantity One software (Bio-Rad) and normalized to β-actin expression. The following primary antibodies were used: anti-TNF-α (1:500), anti-cleaved IL-1β (1:500), and anti-NFκB p65 (Santa Cruz, sc-398442, 1:500).
3.4. The attenuation of POCD by compound K occur via LXR activation Compared with the model group, treatment with 10 mg/kg compound K, but not atorvastatin, attenuated symptoms of cognitive dysfunction after partial hepatectomy in aged mice (Fig. 4A). Treatment with atorvastatin calcium decreased both the levels of serum TNF-α (Fig. 4B) and LDL (Fig. 4C). Treatment with compound K decreased levels of serum TNF-α, IL-1β (Fig. 4B), and LDL (Fig. 4C) and also increased levels of HDL (Fig. 4C). Compared with the model group, treatment with 10 mg/kg compound K, but not atorvastatin, decreased levels of TNF-α and IL-1β in the hippocampus (Fig. 4D). There were significant differences in mRNA levels of ABCA1, ABCG1, and ApoE in hippocampal tissues between the compound K only group and the model group (Fig. 4E). Treatment with GGPP reversed all of effects induced by compound K.
2.8. Statistical analysis All data were expressed as the mean ± SD. Histograms methods was used to evaluate the assumptions of the data. Comparisons between groups were conducted using one-way analysis of variance (ANOVA) plus a post-hoc least significant difference (LSD) test. All analyses were performed using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA). Values of p < 0.05 were considered to be statistically significant.
3. Results 3.1. Compound K attenuated transient POCD caused by partial hepatectomy in aged mice
4. Discussion
The MWM tests were used to determine cognitive impairment induced by partial hepatectomy. Mice in the model group exhibited prolonged escape latency, shortened original quadrant time, and decreased frequency of crossing at both days 10 and 14 as compared with the control group (p < 0.05). These results indicate that spatial memory was significantly impaired after the partial hepatectomy. Treatment with compound K (10, 30 mg/kg) improved symptoms of cognitive dysfunction on all of the MWM tests conducted, when compared with mice in the model group (Fig. 1). The pharmacological effect of compound K was shown to be dose-dependent. Compared with control group, administration of 10 mg/kg compound K did not affect the spatial memory significantly.
During recent years, POCD has become a growing concern for surgical doctors and their support teams. Dysfunctions in short-term and long-term memory, mood, and consciousness can be detected days to weeks after surgical operations and can persist for weeks to months [27]. Aging is well known as a major risk factor of POCD. Compared with young and middle-aged people, a higher ratio of elderly patients suffers from POCD; cognitive dysfunction lasts three months in 14% of patients over sixty years old and symptoms are independent of both the type of operation and anesthetic [28,29]. The pathological mechanism of POCD is not yet completely understood, therefore the therapies are limited. In addition to exclusion of any other disease associated with organic psychosyndrome, and based on the symptoms, the present treatments of POCD are limited to providing sufficient ventilation and oxygenation, hemodynamic support, pain control, promotion of normal circadian rhythms, and maintenance of electrolyte balance [30,31]. Studies have indicated important roles of systemic and local inflammatory cytokines in the presentation of POCD. Our previous study reported hydrogen-rich saline could attenuate the symptoms of POCD via inflammation inhibition [25] and the treatment with thalidomide could attenuate the spatial memory deficit in aged rats after laparotomy by reducing TNF-α and IL-1β levels [32]. These results indicated that anti-inflammatory treatment might be a potential effective method to attenuate POCD. Based on our previous study [10] on the LXRα activation of compound K (310 mg/kg) in mice, and Seo’s [33] and Hou’s [34] studies on the neuroprotective, hippocampal neurogenesis and
3.2. Compound K modulated systemic inflammatory cytokines and serum lipids of aged mice following partial hepatectomy There was no significant difference in serum glucose levels (Fig. 2A) or body weight (Fig. 2B) among the groups. Serum TNF-α and IL-1β levels were significantly increased after partial hepatectomy. Treatment with compound K resulted in decreased levels of cytokines in a dosedependent manner (Fig. 2C). Compared with mice in the model group, serum LDL-C levels were decreased in mice after the administration of compound K (10, 30 mg/kg), while HDL-C levels were significantly increased (Fig. 2D). 7
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
Author contributions
cognition-enhancing effects of compound K (5, 10 mg/kg) in mice, we explored the effects of compound K from 3 mg/kg to 30 mg/kg on POCD in mice. The present study showed that compound K could significantly attenuate the memory dysfunction of aged mice after partial hepatectomy in all MWM tests in a dose-dependent manner; our previous study indicated that compound K was a novel agonist of LXRα [35]. Therefore, we hypothesized that the role of compound K might be influence activation of LXRα. Here, we report that treatment with GGPP reverses the effects of compound K, thus confirming that the pharmacological effects of compound K are dependent on LXRα activation. LXRα is a regulator of reverse cholesterol transport. Activation of LXRα up-regulates expression of the downstream genes such as ABCA1, ABCG1, and ApoE and induces multiple biological alterations including the regulation of serum lipids and inhibition of inflammation [36]. To explore whether serum lipid alteration is involved in the mechanism by which compound K influences POCD, we used atorvastatin calcium as a control. Compared with the model group, treatment with atorvastatin calcium resulted in decreased levels of LDL and TC, as well as serum TNF-α. However, there was no significant difference in MWM tests results between mice in the atorvastatin calcium group and the model group. These results indicate that serum lipid levels do not play an important role in POCD symptoms. Therefore, we can conclude that the effect of serum lipid modulation by compound K did not contribute to the attenuation of POCD. In addition, our results show that systemic cytokines levels were also independent of POCD presentation. This result is consistent with clinical research, indicating that systemic noninfective inflammation is not associated with the development of POCD during the early post-operative period in elderly patients [37]. In the immunohistochemistry analysis, levels of TNF-α and IL-1β in hippocampus tissue exhibited a direct relationship to MWM scores. Treatment with compound K could decrease levels of TNF-α and IL-1β in hippocampal tissue in a dose-dependent manner. A previous study indicated that LXR agonists could alleviate brain inflammation by acting on upregulation of ABCA1 expression in neurons, astrocytes, and microglia [21], which is consistent with our results. Therefore, all these results indicate that the anti-inflammatory effect in local hippocampus tissue plays a key role in the elucidation of the mechanism by which compound K on influences POCD. Increased levels genes downstream of LXRα after hepatectomy indicate that activation of the LXRα pathway might be a protective reaction, which was not strong enough to completely attenuate the inflammation. Compound K, as an agonist, could activate the anti-inflammatory pathway, thereby attenuating the symptoms of POCD. Our previous study demonstrated that compound K was a novel agonist of LXRα without serious side effects [10,35] such as lipid deposition in liver, which are induced by general LXRα activators currently in use including GW3965 and T0901317. Furthermore, other studies have shown that compound K can attenuate fatty liver in rats [38]. The safety of compound K has been confirmed by several reports [39,40]. Therefore, the present study demonstrates a potential therapeutic role for compound K in POCD.
Y. Tian conceived and designed the experiments; Q. Liu and L. Liu performed the in vivo experiments, including operation and samples collection; H. Liu performed the cell experiments; C. Wang contributed Real-time PCR; J. Jiang and Y. Jia analyzed the data; S. Guo contributed MWM tests; Y. Tian, Y. Jia and Q. Liu wrote the paper. All authors have revised the manuscript and given final approval to the version to be published. Declaration of Competing Interest The authors have declared that no conflict of interest exists. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109400. References [1] A. Rudra, S. Chatterjee, J. Kirtania, S. Sengupta, G. Moitra, S. Sirohia, et al., Postoperative delirium, Indian J. Crit. Care Med. 10 (2006) 235–240, https://doi. org/10.4103/0972-5229.29842. [2] C.C. Price, C.W. Garvan, T.G. Monk, Type and severity of cognitive decline in older adults after noncardiac surgery, Anesthesiology 108 (2008) 8–17, https://doi.org/ 10.1097/01.anes.0000296072.02527.18. [3] K.R. Murrin, T.M. Nagarajan, Hyperventilation and psychometric testing. A preliminary study, Anaesthesia 29 (1974) 50–58, https://doi.org/10.1111/j.13652044.1974.tb00582.x. [4] J.T. Moller, P. Cluitmans, L.S. Rasmussen, P. Houx, H. Rasmussen, J. Canet, et al., Long-term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International study of post-operative cognitive dysfunction, Lancet. 351 (1998) 857–861, https://doi.org/10.1016/S0140-6736(97)07382-0. [5] P.A. Barber, S. Hach, L.J. Tippett, L. Ross, A.F. Merry, P. Milsom, Cerebral ischemic lesions on diffusion weighted imaging are associated with neurocognitive decline after cardiac surgery, Stroke 39 (2008) 1427–1433, https://doi.org/10.1161/ STROKEAHA.107.50298. [6] B. Jungwirth, K. Kellermann, M. Qing, G.B. Mackensen, M. Blobner, E.F. Kochs, Cerebral tumor necrosis factor α expression and long-term neurocognitive performance after cardiopulmonary bypass in rats, J. Thorac. Cardiovasc. Surg. 138 (2009) 1002–1007, https://doi.org/10.1016/j.jtcvs.2009.06.022. [7] V. Tomic, S. Russwurm, E. Möller, R.A. Claus, M. Blaess, F. Brunkhorst, et al., Transcriptomic and proteomic patterns of systemic inflammation in on-pump and off-pump coronary artery bypass grafting, Circulation. 112 (2005) 2912–2920, https://doi.org/10.1161/CIRCULATIONAHA.104.531152. [8] Y. Ding, C. Shi, L. Chen, P. Ma, K. Li, J. Jin, et al., Effects of andrographolide on postoperative cognitive dysfunction and the association with NF-κB/MAPK pathway, Oncol. Lett. 14 (2017) 7367–7373, https://doi.org/10.3892/ol.2017. 7088. [9] J.A. Clemens, D.T. Stephenson, E.B. Smalstig, E.P. Dixon, S.P. Little, Global ischemia activates nuclear factorb in forebrain neurons of rats, Stroke. 28 (1997) 1073–1080, https://doi.org/10.1161/01.STR.28.5.1073. [10] L. Zhou, Y. Zheng, Z. Li, L. Bao, Y. Dou, Y. Tang, et al., Compound K attenuates the development of atherosclerosis in ApoE-/- mice via LXRα activation, Int. J. Mol. Sci. 17 (2016) 1054, https://doi.org/10.3390/ijms17071054. [11] S. Kang, M.H. Siddiqi, S.J. Yoon, S. Ahn, H.Y. Noh, N.S. Kumar, et al., Therapeutic potential of compound K as an IKK inhibitor with implications for osteoarthritis prevention: an in silico and in vitro study, In Vitro Cell. Dev. Biol. Anim. 52 (2016) 895–905, https://doi.org/10.1007/s11626-016-0062-9. [12] R. Wang, M. Zhang, S. Hu, K. Liu, Y. Tai, J. Tao, et al., Ginsenoside metabolite compound-K regulates macrophage function through inhibition of β-arrestin2, Biomed. Pharmacother. 115 (2019) 108909, , https://doi.org/10.1016/j.biopha. 2019.108909. [13] S. Lee, M.C. Kwon, J.P. Jang, J.K. Sohng, H.J. Jung, The ginsenoside metabolite compound K inhibits growth, migration and stemness of glioblastoma cells, Int. J. Oncol. 51 (2017) 414–424, https://doi.org/10.3892/ijo.2017.405. [14] C. Hu, G. Song, B. Zhang, Z. Liu, R. Chen, H. Zhang, et al., Intestinal metabolite compound K of panaxoside inhibits the growth of gastric carcinoma by augmenting apoptosis via Bid-mediated mitochondrial pathway, J. Cell. Mol. Med. 16 (2012) 96–106, https://doi.org/10.1111/j.1582-4934.2011.01278.x. [15] Y.C. Hwang, D.H. Oh, M.C. Choi, S.Y. Lee, K.J. Ahn, H.Y. Chung, et al., Compound K attenuates glucose intolerance and hepatic steatosis through AMPK-dependent pathways in type 2 diabetic OLETF rats, Korean J. Intern. Med. (2017), https://doi. org/10.3904/kjim.2015.208. [16] W. Chen, J. Wang, Y. Luo, T. Wang, X. Li, A. Li, et al., Ginsenoside Rb1 and compound K improve insulin signaling and inhibit ER stress-associated NLRP3 inflammasome activation in adipose tissue, J. Ginseng Res. 40 (2016) 351–358. [17] E. Kim, D. Kim, S. Yoo, Y.H. Hong, S.Y. Han, S. Jeong, et al., The skin protective
5. Conclusions Overall, the present work sheds light on the alleviation of POCD by compound K via inhibition of the local inflammatory response in hippocampal tissue; furthermore, this mechanism is likely to be associated with LXRα activation. These findings suggest further evaluation of compound K as a potential effective modulator for POCD is needed.
Funding This work was supported by the National Natural Science Foundation of China (No. 81701075). 8
Biomedicine & Pharmacotherapy 119 (2019) 109400
Q. Liu, et al.
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
19434.1e. [29] L. Evered, D.A. Scott, B. Silbert, P. Maruff, Postoperative cognitive dysfunction is independent of type of surgery and anesthetic, Anesth. Analg. 112 (2011) 1179–1185, https://doi.org/10.1213/ANE.0b013e318215217e. [30] W. Wang, Y. Wang, H. Wu, L. Lei, S. Xu, X. Shen, et al., Postoperative cognitive dysfunction: current developments in mechanism and prevention, Med. Sci. Monit. 20 (2014) 1908–1912, https://doi.org/10.12659/MSM.892485. [31] M. Pappa, N. Theodosiadis, A. Tsounis, P. Sarafis, Pathogenesis and treatment of post-operative cognitive dysfunction, Electron. Physician 9 (2017) 3768–3775, https://doi.org/10.19082/3768. [32] P. Guo, S.P. Hu, Thalidomide alleviates postoperative pain and spatial memory deficit in aged rats, Biomed. Pharmacother. 95 (2017) 583–588, https://doi.org/10. 1016/j.biopha.2017.08.114. [33] J.Y. Seo, S.H. Ju, J. Oh, S.K. Lee, J.S. Kim, Neuroprotective and cognition-enhancing effects of compound K isolated from red ginseng, J. Agric. Food Chem. 64 (2016) 2855–2864, https://doi.org/10.1021/acs.jafc.5b05789. [34] J.G. Hou, J.J. Xue, M.R. Lee, M.Q. Sun, X.H. Zhao, Y.N. Zheng, et al., Compound K is able to ameliorate the impaired cognitive function and hippocampal neurogenesis following chemotherapy treatment, Biochem. Biophys. Res. Commun. 436 (2013) 104–109, https://doi.org/10.1016/j.bbrc.2013.05.087. [35] Y. Huang, H. Liu, Y. Zhang, J. Li, C. Wang, L. Zhou, et al., Synthesis and biological evaluation of ginsenoside compound K derivatives as a novel class of LXRα activator, Molecules. 22 (2017) 1232, https://doi.org/10.3390/molecules22071232. [36] S.S. Im, T.F. Osborne, Liver X receptors in atherosclerosis and inflammation, Circ. Res. 108 (2011) 996–1001, https://doi.org/10.1161/CIRCRESAHA.110.226878. [37] E. Nemeth, K. Vig, K. Racz, K.B. Koritsanszky, K.I. Ronkay, F.P. Hamvas, et al., Influence of the postoperative inflammatory response on cognitive decline in elderly patients undergoing on-pump cardiac surgery: a controlled, prospective observational study, BMC Anesthesiol. 17 (2017) 113, https://doi.org/10.1186/ s12871-017-0408-1. [38] X.J. Chen, W.J. Liu, M.L. Wen, H. Liang, S.M. Wu, Y.Z. Zhu, et al., Ameliorative effects of Compound K and ginsenoside Rh1 on non-alcoholic fatty liver disease in rats, Sci. Rep. 7 (2017) 41144, https://doi.org/10.1038/srep41144. [39] Y.L. Gao, Z.F. Liu, C.M. Li, J.Y. Shen, H.X. Yin, G.S. Li, Subchronic toxicity studies with ginsenoside compound K delivered to dogs via intravenous administration, Food Chem. Toxicol. 49 (2011) 1857–1862, https://doi.org/10.1016/j.fct.2011. 05.00. [40] L. Chen, L. Zhou, J. Huang, Y. Wang, G. Yang, Z. Tan, et al., Single- and multipledose trials to determine the pharmacokinetics, safety, tolerability, and sex effect of oral ginsenoside compound k in healthy Chinese volunteers, Front. Pharmacol. 8 (2018) 965, https://doi.org/10.3389/fphar.2017.00965.
effects of compound K, a metabolite of ginsenoside Rb1 from Panax ginseng, J. Ginseng Res. 42 (2018) 218–224. J. Oh, J.S. Kim, Compound K derived from ginseng: neuroprotection and cognitive improvement, Food Funct. 7 (2016) 4506–4515, https://doi.org/10.1039/ c6fo01077f. J. Chen, H. Wu, Q. Wang, Y. Chang, K. Liu, W. Wei, Ginsenoside metabolite Compound K suppresses T-cell priming via modulation of dendritic cell trafficking and costimulatory signals, resulting in alleviation of collagen-induced arthritis, J. Pharmacol. Exp. Ther. 353 (2015) 71–79, https://doi.org/10.1124/jpet.114. 220665. J. Chen, M. Si, Y. Wang, L. Liu, Y. Zhang, A. Zhou, et al., Ginsenoside metabolite compound K exerts anti-inflammatory and analgesic effects via downregulating COX2, Inflammopharmacology. 27 (2019) 157–166, https://doi.org/10.1007/ s10787-018-0504-y. I. Lefterov, A. Bookout, Z. Wang, M. Staufenbiel, D. Mangelsdorf, R. Koldamova, Expression profiling in APP23 mouse brain: inhibition of Abeta amyloidosis and inflammation in response to LXR agonist treatment, Mol. Neurodegener. 2 (2007) 20, https://doi.org/10.1186/1750-1326-2-20. A. Archer, E. Stolarczyk, M.L. Doria, L. Helguero, R. Domingues, J.K. Howard, et al., LXR activation by GW3965 alters fat tissue distribution and adipose tissue inflammation in ob/ob female mice, J. Lipid Res. 54 (2013) 1300–1311, https://doi. org/10.1194/jlr.M033977. E. Vucic, C. Calcagno, S.D. Dickson, J.H. Rudd, K. Hayashi, J. Bucerius, et al., Regression of inflammation in atherosclerosis by the LXR agonist R211945: a noninvasive assessment and comparison with atorvastatin, JACC Cardiovasc. Imaging 5 (2012) 819–828, https://doi.org/10.1016/j.jcmg.2011.11.025. C. Hu, A.J. Lau, R. Wang, T.K.H. Chang, Comparative analysis of ginsenosides in human glucocorticoid receptor binding, transactivation, and transrepression, Eur. J. Pharmacol. 815 (2017) 501–511, https://doi.org/10.1016/j.ejphar.2017.10.019. Y. Tian, S. Guo, Y. Zhang, Y. Xu, P. Zhao, X. Zhao, Effects of hydrogen-rich saline on hepatectomy-induced postoperative cognitive dysfunction in old mice, Mol. Neurobiol. 54 (2017) 2579–2584, https://doi.org/10.1007/s12035-016-9825-2. Y. Tian, S. Guo, L. Ma, Y. Wang, Y. Xu, P. Zhao, Sevoflurane aggregates cognitive dysfunction and hippocampal oxidative stress induced by β-amyloid in rats Author links open overlay panel, Life Sci. 143 (2015) 194–201, https://doi.org/10.1016/j. lfs.2015.11.002. L. Krenk, L.S. Rasmussen, H. Kehlet, New insights into the pathophysiology of postoperative cognitive dysfunction, Acta Anaesthesiol. Scand. 54 (2010) 951–956, https://doi.org/10.1111/j.1399-6576.2010.02268. T.G. Monk, B.C. Weldon, C.W. Garvan, D.E. Dede, M.T. van der Aa, K.M. Heilman, et al., Predictors of cognitive dysfunction after major noncardiac surgery, Anesthesiology. 108 (2008) 18–30, https://doi.org/10.1097/01.anes.0000296071.
9