Ononin, a natural isoflavone glycoside, alleviates postoperative cognitive dysfunction in aged mice by regulating neuroinflammation and oxidative stress

doi:10.21203/rs.3.rs-4972022/v1

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Ononin, a natural isoflavone glycoside, alleviates postoperative cognitive dysfunction in aged mice by regulating neuroinflammation and oxidative stress

https://doi.org/10.21203/rs.3.rs-4972022/v1

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Postoperative cognitive dysfunction (POCD) is a common and serious neurological complication. Currently, there is no effective clinical prevention and treatment for POCD. Ononin has been confirmed to exhibit potent neuroprotective effects in many diseases. This study aimed to investigate whether ononin could exert a neuroprotective role against POCD. The animal model of POCD was established in 18-month-old aged mice with unilateral nephrectomy. Ononin (30 mg/kg) was administered intraperitoneally to aged mice 15 min before surgery. On postoperative day 3, the Morris water maze and open field tests were used to assess the changes in cognitive function. Western blotting and immunofluorescence staining were employed to examine the hippocampal levels of Iba1 and microglial activation on postoperative day 3, respectively. An enzyme-linked immunosorbent assay was applied to gauge the expression of hippocampal IL-1β, IL-6, and TNF-α on days 1 and 3 postsurgery. To reflect the oxidative stress status, the levels of hippocampal malondialdehyde (MDA) and superoxide dismutase (SOD) activity were detected using the corresponding assay kits on postoperative days 1 and 3. We found that anesthesia/surgery induced overt memory deficits in aged mice. Conversely, ononin pretreatment significantly rescued the cognitive impairment. Mechanically, anesthesia/surgery triggered acute increases in hippocampal IL-1β, IL-6, TNF-α, Iba1, and MDA, paralleled by a decline in SOD activity. This phenomenon was also partially reversed by ononin. Our findings provide evidence that ononin may ameliorate anesthesia/surgery-induced cognitive deficits through its anti-inflammatory and antioxidant effects, which could be a novel preventive therapeutic strategy for POCD in elderly patients.

Ononin

Postoperative cognitive dysfunction

Aging

Neuroinflammation

Oxidative distress

Postoperative cognitive dysfunction (POCD) is a serious neurological complication following major surgery, especially in aged patients [1]. The reported incidence of POCD ranges between 15–60% [2, 3]. It is characterized by a decrease in learning, memory, concentration, or executive function following operation [4]. Patients with POCD often exhibit reduced quality of life, prolonged hospital stays, and increased cost of hospitalization [5, 6]. Additionally, POCD may result in an increase in surgical morbidity and mortality [7]. Notably, cognitive impairment after anesthesia/surgical stress may not be transient, which could last for several years or even longer, potentially increasing the prevalence of Alzheimer's disease (AD) [8]. As elderly people are expected to be the largest group undergoing surgery, their risk of developing POCD will increase significantly. Therefore, there is an urgent need to find ways to prevent the progression of POCD. Unfortunately, few effective methods have been excavated for the prevention and treatment of POCD to date.

Neuroinflammation [5], synaptic plasticity dysfunction [9], amyloid-β (Aβ) deposition [10], mitochondrial damage [11], and neuronal apoptosis [12] have been proved to be implicated in the mechanisms of POCD. Nevertheless, an increasing number of researches have shown that among these mechanisms, neuroinflammatory reaction was the central link and played a pivotal role in the onset and development of POCD [13]. Aseptic peripheral surgery or trauma could trigger immunological reactions, resulting in systemic release of proinflammatory cytokines [14]. Preclinical studies revealed that tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), three major pro-inflammatory factors, could cross the impaired blood-brain barrier (BBB) and enter the central nervous system, ultimately causing neuroinflammation and oxidative distress [15, 16]. Further, microglia are activated, leading to the deterioration of neuroinflammation and oxidative damage [17]. Existing evidence suggests that the interaction between oxygen free radicals and pro-inflammatory factors may exacerbate POCD [18, 19]. Interventions that attenuate neuroinflammation and reactive oxygen species (ROS) could protect against cognitive impairment following surgery in aged mice [17, 20, 21]. Based on the above studies, treatments devoted to attenuating neuroinflammation and oxidative distress hold great potential in the prevention and treatment of POCD.

Ononin (Fig. 1a), a representative isofavone component, is found in all sorts of traditional Chinese medicines, such as Glycyrrhiza uralensis Fisch. ex DC. (Fabaceae), Pueraria montana (Lour.) Merr. (Fabaceae), Astragalus trimensis L. (Fabaceae), and Hedysarum spp. L. (Fabaceae). Previous studies have demonstrated that ononin possesses pleiotropic biological activities, with the most focused on its anti-inflammatory and antioxidant effects [22–24]. Chen et al. found that ononin treatment exhibited potent neuroprotective effects in AD through inhibiting neuroinflammation and oxidative distress in rodents [23]. In addition, Fu and colleagues uncovered that a structural analogue of ononin, calycosin-7-O-β-D-glucoside could also afford neuroprotective effects in the middle cerebral artery occlusion (MCAO) ischemia-reperfusion model in rats via regulating the NO/cav-1/MMPs pathway [25]. Of note, recent evidence suggests that ononin could ameliorate depression-like behaviors in rats via regulating BDNF-TrkB-CREB signaling [26]. Nevertheless, the therapeutic effect of ononin in POCD remains largely unclear. Consequently, the present study was conducted to determine whether ononin treatment could alleviate anesthesia/surgery-induced cognitive deficits in elderly mice via suppression of neuroinflammation and oxidative distress.

Animals

18-month-old Wild-type C57BL/6 male mice, weighing between 30-36g, were purchased from Sippe-Bk Lab Animal Company (Shanghai, China). Five individuals per cage were housed in a specific-pathogen-free (SPF) room (23 ± 1 ℃) and allowed to freely obtain food and water under 12–12 h light–dark cycles. The humidity of the SPF room was kept at 60 ± 5%. Before the experiment, mice were permitted for adaptation of the laboratory circumstance for 7 days. Our study was approved by the Ethics Committee of North Sichuan Medical College Institutional (dated: 08/06/2022). All procedures were executed in line with the “Guide for the Care and Use of Laboratory Animals” published by the NIH.

Experimental design

Animals were randomly (Computer-based randomization) allocated into the following groups (n = 18 per group): control, surgery, ononin, and surgery + ononin groups (surgery + ononin). Ononin (Sigma-Aldrich, USA) was dissolved in saline containing 1% DMSO (Solarbio, China). The ononin and the surgery + ononin groups received ononin by intraperitoneal injection (30 mg/kg) [25] 15 min before onset of surgery. At the corresponding time point, the other two groups were administrated to an equal volume of saline containing 1% DMSO. The schematic diagram of the experimental design is described in Fig. 1b.

Animal model of POCD

Unilateral nephrectomy is a well-established method for the development of a mouse POCD model [21, 27]. Thus, as our previously described approach [28], unilateral nephrectomy was carried out to generate a POCD model in aged mice. Briefly, mice were inducted with 4% sevoflurane through a conical mask (2% in oxygen), followed by 2%-2.5% for maintenance. Body temperature during the procedure was kept between 36.5 ℃ to 37.5 ℃ utilizing a heating pad. Then, we carefully shaved off the hair in the surgical area and thoroughly cleaned it with 75% ethanol. A transverse incision of approximately 1.5 cm was made and the left kidney was exteriorized and carefully resected. For postoperative analgesia, a subcutaneous injection of 200 ul ropivacaine (0.2%) was used. The researchers, who were aware of the detailed grouping of the mice, took responsibility for collecting experimental samples, but the evaluators of the experimental results were blinded to the allocation of the mice groups. The entire surgical procedure took approximately 40 min. Mice that did not undergo surgery received only a 1:1 mixture of air and 100% oxygen treatment for the same period of time.

Open field test

Open field test (OFT) is used to estimate the autonomous activity and spatial exploration in rodents. OFT was conducted on day 3 postoperatively based on our previous study [12] with slight modifications. Mouse was placed gently into the center (50% of the total area [29]) of the OFT device (50 × 30 × 25 cm³), and allowed free exploration for 5 min. A video camera was employed for automatically recording the exploratory locomotor activities. The total travel distance, mean velocity, rearing activity and duration in the center were analyzed by means of a maze scan software (Top-view v.3.0, Cleversys, USA). After each test, the OFT apparatus was cleaned with 75% alcohol.

Morris water maze test

Morris water maze (MWM) is a classical and reliable means for the evaluation of spatial learning and memory in rodents [30]. As described previously [31], the MWM test was executed with minor modifications. In brief, prior to the start of the test, mice were transported into the test room for habituation at least 30 min. A circular tank pool with a 50 cm depth and 120 cm diameter was filled to a depth of 30 cm with opaque water made by adding white and non-toxic dye. We maintained the water temperature at 23 ± 1°C and divided the tank pool into an average of 4 quadrants. A dim light was put into the test room, and four different shapes of visual cues were placed above each quadrant of the pool to assist the mice to navigate and locate the submerged platform. A platform with a diameter of 10 cm was hidden 1 cm below the water surface and placed in the middle of one quadrant. The platform remained unchanged throughout the training phase and was removed during the probe testing. A video camera was installed above the pool and connected to the maze scan software (Cleversys, USA) for tracing and analyzing the animal’s movement. In the training session (acquisition), 4 consecutive trials per day were conducted on each mouse for 5 days before the operation (Fig. 1b). Each mouse facing the tank wall was put into the water in the desired starting position. The time limit for mice to find and climb onto the platform was 60 s. If the mice could not climb onto the platform in the allotted time, we gently placed them on the platform and let them stay for 20 s. During the acquisition, the swimming speed and the latency to the platform were calculated for each day. On postoperative day 3, the platform was removed. In the opposite quadrant, mice were released and tested for 60 s in the probe testing. For this session, the latency to the previous platform, the percentage of time in the platform quadrant, and the entries across the previous platform were recorded and analyzed.

Brain tissue preparation

Mouse hippocampal tissues were harvested on days 1 and 3 after surgery. On the 1st day postsurgery, six mice from each group were anesthetized with a lethal dose of sodium pentobarbital (100 mg/kg, intraperitoneal injection), and then transcardially perfused with ice-cold 0.9% saline. The brain tissue was swiftly extracted with ophthalmic scissors and forceps. Subsequently, the hippocampus was carefully isolated and transferred into a 1.5 mL freezing EP tube, followed by stored at -80 ◦C in a refrigerator for subsequent assays, including enzyme linked immunosorbent assay (ELISA) and anti-oxidative enzyme activity assay, respectively. On day 3 after surgery, the remaining mice (except for those mentioned below for western blot analysis and immunohistochemistry) were euthanized after the end of behavioral test using the same method. The hippocampus was collected to examine the levels of inflammatory factors and oxidative stress response.

Oxidative distress determination

According to the manufacturer's instructions from Nanjing Jiancheng Bioengineering Institute, the anti-oxidant enzymes superoxide dismutase (SOD) assay kit and the oxidative product malondialdehyde (MDA) assay kit (Nanjing, China) were utilized to measure hippocampal SOD activity and MDA levels. A microplate reader (VirusScan LUX, USA) was employed to detect the corresponding OD values (MDA at 532 nm, SOD at 450 nm). Based on the formula in the kit manual, MDA levels (nmol/mg protein) and SOD activity (U/mg protein) were calculated.

Determination of inflammatory markers.

The hippocampus was obtained on days 1 and 3 after unilateral nephrectomy. We employed ELISA kits (Fine Test, #EM0183, #EM0109 and #EM0121, China) to determine the levels of TNF-α, IL-6, and IL-1β in the light of the manufacturer’s protocols.

Immunofluorescence analysis

Ionized calcium binding adapter molecule 1 (Iba1), an important marker of microglia, was immunofluorescence stained in accordance with the antibody manufacturer to investigate microglial morphology. Briefly, mouse was deeply anesthetized, and then subjected to transcardiac perfusion with 25 ml of 0.9% saline, followed by 20 ml of 4% paraformaldehyde (PFA). After that, the brain was removed, fixed by 4% PFA for 24 h, and embedded in the thick paraffin. Then, the brain was dissected at a thickness of 3 µm. The slices were subjected to dewaxing, rehydration, and antigen repair in sequence. Thereafter, donkey serum (10%) (Solarbio, China) was utilized to block the non-specific binding sites at 37°C for 1 h. The slices were then incubated with the primary antibody (rabbit anti-Iba1, #ab178846, Abcam, 1:100, UK) overnight at 4°C. Subsequently, the slides were briefly washed with phosphate balanced solution three times for 5 min, followed by incubation with secondary antibody (Abcam, #ab150077, 1:200, UK) for 1 h at room temperature. Then, DAPI was applied for counterstaining nuclei for 5 min. Through an Olympus confocal BX-60 imaging system (Tokyo, Japan), the images of hippocampal CA1 region in aged mice were obtained at 400× magnification. The number and average fluorescence intensity of Iba1 positive cells in the hippocampal CA1 region were analyzed by means of Image J (NIH, USA).

Western blot analysis

Western blotting was conducted based on our previous research [12]. In brief, equal amounts (100 µg of protein) of the sample were separated using 15% SDS-PAGE gels, and then transferred to activated PVDF membranes (0.45 µm) (Millipore, United States). Afterwards, at room temperature, the membranes were blocked for 1 h with 5% non-fat milk solution, and incubated overnight at 4°C by the corresponding primary antibodies against Iba1 (Abcam, #ab178846, 1:1000, UK) and GAPDH (Proteintech, #10494-1-AP, 1:3000, China). After three washes with TBST, the membranes were incubated for another 1 h at room temperature with HRP-conjugated secondary antibodies (Zsbio, #ZB-2306, 1:3000, China). The targeted protein band was visualized through employing an Enhanced Chemiluminescence Kit (Millipore, #WBKLS0100, United States). Bands were quantified using Image J 8.0.

Data analysis and statistics

All data were presented as mean ± SEM. Prism 9.0 software (GraphPad software, USA) was applied to perform statistical analyses. Normality of the experimental data was evaluated by the Shapiro-Wilk test. The data of MWM were analyzed by two-way analysis of variance (ANOVA) followed by the Tukey post hoc test for repeated measures. For other data, such as open field test, quantification of immunoreactivity, and normalized band intensities in western blot, we used one-way ANOVA followed by the Tukey post hoc test for the assessment of statistical significance. Differences with P < 0.05 were considered statistically significant.

Unilateral nephrectomy imposed a negligible effect on the spontaneous locomotor activities

We employed the OFT for the evaluation of spontaneous locomotor activities of aged mice on day 3 postoperatively. Our results indicated that there was little difference between groups (P > 0.05) with regard to total travel distance (Fig. 2a), mean velocity (Fig. 2b), rearing activity (Fig. 2c), and duration in the center (Fig. 2d-e). These data suggest that unilateral nephrectomy had negligible influences on the spontaneous locomotor activities, and the cognitive dysfunction in surgery-treated mice was unlikely to be due to impaired spontaneous locomotor ability.

Ononin prophylaxis preserved learning and memory following unilateral nephrectomy

In the acquisition of the MWM test, the spatial learning and memory abilities of elderly mice improved after training over time, and no differences were detected in the average swimming speed and escape latency between groups (P > 0.05, Fig. 3a-c). During the probe testing, there was also little difference among the four groups in terms of average swimming speed (P > 0.05, Fig. 4a). However, the number of crossing platforms and the percentage of time spent in the target quadrant were significantly reduced in surgery-treated mice on day 3 postoperation when compared to the controls (P < 0.001, Fig. 4b, c). On the contrary, the latency to the previous platform increased markedly in the surgery group when compared to the control group (P < 0.001, Fig. 4d, e), suggesting that unilateral nephrectomy may cause learning and memory deficits in elderly mice. Importantly, anesthesia/surgery-induced cognitive decline was notably ameliorated by ononin pretreatment (P < 0.05, Fig. 4b-e).

Ononin attenuated anesthesia/surgery-triggered increases in hippocampal pro-inflammatory cytokines

Neuroinflammation related to surgical trauma is a critical pathogenic element for the development of POCD [32]. Thus, to determine whether the therapeutic role of ononin on cognitive defect was linked to the reduced neuroinflammation, we measured the content of pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) in the hippocampus, a vital brain region associated with cognition [33]. As shown in Fig. 5a-f, on postoperative days 1 and 3, these three cytokines in the hippocampal tissues of surgery-treated mice were considerably elevated (P < 0.001), while this increase was significantly reversed at both time points by application of ononin (P < 0.05). These findings verified that ononin pretreatment could dampen anesthesia/surgery-induced hippocampal inflammation.

Ononin reversed anesthesia/surgery-induced hippocampal microglial activation in aged mice

Activated microglia exert a crucial role in surgery-elicited neuroinflammation and neuronal damage [34]. Therefore, to investigate hippocampal microglial activation in aged mice, we utilized immunofluorescence staining and western blotting to examine the levels of Iba1, a reliable cytoplasmic microglial marker with a strong signal. Our data showed that the number and average fluorescence intensity of Iba1-positive cells in the hippocampal CA1 region were obviously increased in the surgery-treated mice when compared to the controls (P < 0.001, Fig. 6a-c). Meanwhile, the Iba1-positive cell bodies displayed marked hypertrophy after surgery (Fig. 6a). We also found that the protein levels of Iba1 were dramatically enhanced in the surgery group when compared to the control group (P < 0.001, Fig. 6d, e). By contrast, ononin pretreatment significantly attenuated the anesthesia/surgery-induced alteration of Iba1and microglia (P < 0.05, Fig. 6a-e).

Ononin ameliorated anesthesia/surgery-elicited hippocampal oxidative distress response in aged mice

The excessive mitochondrial oxidative stress response also contributed to increased susceptibility to POCD [35], we therefore determined whether ononin could exert neuroprotective effects by alleviating oxidative distress. As expected, SOD activity in the hippocampus was considerably reduced in the surgery-treated mice on postoperative days 1 and 3 relative to the controls (P < 0.01, Fig. 7a, c). However, the decreased SOD activity after surgery was largely rescued by ononin administration (P < 0.05, Fig. 7a, c). In contrast, MDA levels were greatly elevated on days 1 and 3 postsurgery (P < 0.001, Fig. 7b, d) in the hippocampus of surgery-treated mice when compared with the controls. Likewise, ononin pretreatment partially reversed the post-operative increase in hippocampal MDA levels (P < 0.05, Fig. 7b, d).

In this study, we found that cognitive deficits in elderly mice could be elicited by unilateral nephrectomy on postoperative day 3. Importantly, the effect of anesthesia/surgical stress was notably reversed by ononin pretreatment. Mechanically, we demonstrated that ononin could remarkably attenuate unilateral nephrectomy-induced hippocampal neuroinflammation and oxidative distress in elderly mice. As a result, our findings provide preclinical evidence that ononin may act as a promising therapeutic drug for anesthesia/surgery-triggered cognitive impairment via mitigating hippocampal inflammation and oxidative insults.

Previously, several POCD models in rodents have been established to mimic the condition that patients may suffer from cognitive impairment following surgery, including exploratory laparotomy [36], tibial fracture surgery [37], and excision of important organs under anesthesia [38]. In the current study, unilateral nephrectomy in aged mice was conducted under sevoflurane anesthesia to set up a POCD model. As demonstrated in previous reports [21, 38], we corroborated that this surgical approach could elicit early postoperative cognitive impairment in aged mice. Meanwhile, the aged mice that underwent unilateral nephrectomy did not alter mean velocity and total travel distance relative to the controls in the OFT, indicating that the cognitive deficits in surgery-treated mice were not attributed to the impairment of spontaneous locomotor ability. Also, we found that our protocol had no impact on the kidney function and mortality in aged mice after surgery (data not shown), which is consistent with the previously published literature [39] and in line with clinical practice.

The MWM is a classical approach for the evaluation of hippocampal-dependent learning and memory in rodents [31]. In this study, after 5 consecutive days of MWM training, the aged mice in each group were able to find the platform quickly and possessed similar spatial memory in the MWM before surgery. On day 3 postsurgery, mice exposed to anesthesia/surgery manifested memory deficits, as evidenced by increased latency to the previous platform and decreased percentage of time spent in the target quadrant, as well as a reduced number of crossing platforms compared to the controls. These results are in agreement with previous studies [18, 40]. Of note, pre-operative intervention with ononin significantly reversed the water-maze impairment in aged mice, denoting that ononin could ameliorate cognitive function following anesthesia/surgery.

A growing body of research has demonstrated the crucial role of neuroinflammation in the pathophysiology of POCD [41]. Liu and colleagues [17] found that suppression of hippocampal neuroinflammation by Sirtuin 3 protected elderly mice from anesthesia/surgery-induced cognitive impairment. Likewise, a large number of preclinical studies unveiled that administration of anti-neuroinflammatory drugs, such as dexmedetomidine [18], fluoxetine [42], and omega-3 fatty acids [14], could relieve surgery-triggered neuroinflammation and preserve learning and memory abilities in aged rodents. In addition, it is well-documented that microglia can exert a key role in immune regulation and phagocytosis of dangerous cells or materials [43]. Surgery-evoked elevations of hippocampal pro-inflammatory cytokines can further hyperactivate microglia and exacerbate neuroinflammation [44]. Activated microglia can elicit neurotoxicity, resulting in damage to synapses and neurons [45, 46]. Our results revealed that hippocampal levels of TNF-α, IL-1β, and IL-6 were markedly elevated on days 1 and 3 postsurgery, accompanied by overt activation of Iba1-labelled microglia, which was consistent with previous researches [12, 17]. By contrast, ononin pretreatment attenuated anesthesia/surgery-triggered increases in hippocampal proinflammatory cytokines and reversed microglial activation to some degree. Thus, the improvement of anesthesia/surgery-induced cognitive decline by ononin may be closely related to its anti-neuroinflammatory properties.

Clinical evidence has uncovered that advanced age is one of the main risk factors of developing POCD [47, 48]. As individuals age, oxidative stress in the brain becomes increasingly evident, characterized by increased ROS levels and decreased anti-oxidant enzymes SOD activity [49]. Oxidative imbalances can cause the accumulation of harmful proteins and dysfunctional mitochondria in elderly patients, which is another pivotal factor contributing to POCD [35, 50]. Crucially, oxidative distress and neuroinflammation coexist and interact, further exacerbating anesthesia/surgery-induced cognitive impairment [18, 51]. Accumulating studies have revealed that ononin possessed potent antioxidant effects [52]. For example, a recent study demonstrated that ononin treatment could mitigate cognitive impairment in AD rats elicited by aluminium chloride through suppressing oxidative distress and neuroinflammation [23]. In addition, Yan and colleagues [53] found that ononin could effectively alleviate OGD/R-evoked HT22 cell damage and apoptosis by suppressing oxidative distress. This knowledge inspired us to explore the latent benefits of ononin on oxidative insults and cognitive decline induced by anesthesia/surgery. In concordant with previous researches [16], we unveiled that unilateral nephrectomy could trigger hippocampal oxidative distress in surgery-treated mice, as evidenced by apparently reduced SOD activity and elevated MDA content on days 1 and 3 postsurgery. Not surprisingly, ononin prophylaxis appreciably reversed these changes, which was in accordance with the decreased neuroinflammation in the hippocampus. This coincides with the extensively believed antioxidant characteristics of ononin in several neurological diseases, such as AD [23]. Accordingly, these findings suggest that the neuroprotective role of ononin in POCD may be implicated in its antioxidant effects as well.

In our study, the reason why we selected the dosage of 30 mg/kg ononin and injected it intraperitoneally into aged mice 15 min before surgery was based on the safety and efficacy of ononin application. A previous study [25] showed that a representative component of isoflavones in Astragalus trimensis L. (Fabaceae), calycosin-7-O-β-D-glucoside, which is a structural analogue of ononin, administered intraperitoneally 15 min prior to cerebral ischemia could afford neuroprotective effects in a rat model of MCAO ischemia-reperfusion. Combined with the results of our pilot research (n = 6 in each group, unpublished data), ononin (30 mg/kg) administered intraperitoneally 15 min before surgery was also safe and substantially mitigated cognitive deficits in surgery-treated mice and the accumulation of hippocampal proinflammatory cytokines and MDA levels. Therefore, we finally chose the dosage of 30 mg/kg ononin for this study and injected it intraperitoneally into aged mice 15 min before surgery to further determine whether ononin could play a neuroprotective role against POCD.

It is essential to acknowledge some limitations in our study. First, we only explored the therapeutic potential of ononin in the early stage of POCD, therefore, further studies are needed to probe the role of ononin in long-term cognitive dysfunction after surgery. Second, increasing evidence has demonstrated that anesthesia and/or surgery could cause neuroinflammation and cognitive decline in elderly mice [54, 55]. Referring to many previous research designs [56, 57], we did not set up a “sham surgery” group as well because sham surgery is also a surgical trauma and requires anesthesia. In clinical practice, anesthesia and surgery are almost inseparable, so we mainly investigate the impacts of anesthesia and surgery in a group on postoperative cognitive function. Third, it is well known that there are many biological differences between humans and mice, so intraperitoneal injection of ononin (30 mg/kg) as a model in elderly mice cannot be extrapolated to humans. Fourth, although our results provide evidence that ononin may hinder or postpone the development of POCD, the precise molecular mechanisms remain poorly understood and need further investigation. Finally, to improve clinical relevance and translational value, future research is needed to determine the optimal dosage, administration methods, as well as the timing of ononin application in patients to diminish cognitive dysfunction induced by anesthesia/surgery.

In conclusion, our study demonstrated that ononin pretreatment could ameliorate anesthesia/surgery-induced cognitive deficits in elderly mice, possibly through its anti-inflammatory and antioxidant effects, which provides a valuable theoretical basis for its use in the prevention and treatment of postoperative cognitive impairment.

Author Contributions QBY, JYL, ML, QMP, and CLC designed the experiments. ML, QMP, JL, CLC and SMY performed the experiments. ML, QMP, QBY, CSG and JYL analyzed data. ML, QMP, QLL and MZ wrote the main manuscript text. All authors have reviewed the manuscript.

Acknowledgments The authors thank Zheng Zhang, Li Li and Xiaolin Wang for their technical assistance.

Funding This work was supported by the special cooperation project between Nanchong city and North Sichuan Medical College (Grant No. 22SXQT0026), the Scientific Research and Development Program of North Sichuan Medical College (Grant No. 7500520016), and the Natural Science Foundation of Fujian Province (Grant No. 2021J01385).

Ethics approval All animal experiments were approved by North Sichuan Medical College Institutional Animal Care and Use Committee.

Declaration of competing interest The authors declare no competing interests.

Data availability Upon a reasonable request, all data are available from corresponding authors.

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Ononin, a natural isoflavone glycoside, alleviates postoperative cognitive dysfunction in aged mice by regulating neuroinflammation and oxidative stress

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Abstract

Figures

Introduction

Materials and methods

Animals

Experimental design

Animal model of POCD

Open field test

Morris water maze test

Brain tissue preparation

Oxidative distress determination

Immunofluorescence analysis

Western blot analysis

Data analysis and statistics

Results

Unilateral nephrectomy imposed a negligible effect on the spontaneous locomotor activities

Ononin prophylaxis preserved learning and memory following unilateral nephrectomy

Discussion

Declarations

References

Additional Declarations

Status:

Version 1