Discussion of the prom ising effect of electroacupuncture on cognitive improvement in D-galactose-induced aging rats based on NLRP3-ASC-Caspase-1 signaling pathway

Zhao Fei-yi (趙非一),Zhao Ying-xia (趙英俠), Yan Hai-xia (燕海霞),Xu Hong (許紅),Hong Yu-fang (洪鈺芳),Xu Yan (徐燕)

1Department of Nursing,Schoolof International Medical Technology,ShanghaiSanda University,Shanghai 201209,China

2 School of Basic Medicine,ShanghaiUniversity of Traditional ChineseMedicine,Shanghai201203,China

3 ShanghaiMunicipal Hospital of TCM A ffiliated to ShanghaiUniversity of TraditionalChinese Medicine,Shanghai200071,China

Abstract

Keywords: Acupuncture Therapy; Electroacupuncture; Aging; Cognitive Dysfunction;Behavior, Animal; NLRP3 Protein;Inflammasomes; Rats

Aging is an extremely complex multifactorial biological phenomenon,accompanied by a gradual decline in the physiologic function of organisms, and resulting in molecular,cellular and organic dysfunction[1-2].Brain aging is a critical aspect of the aging process,which is characterized by cognitive decline reflected by learning and memory deficits or impairments that could be the result of oxidative stress[3-4]. Furthermore, cognitive deficit is among the most feared aspects of aging as it may herald dementia,illness or even death[5]. Understanding the changes and underlying molecular mechanisms of aging cerebral tissue is of great significance for the study of aging-associated cognitive decline[6]. This is also one of the research hotspots of geriatrics in recent years.

Accumulating evidence supports the prom ising effects of acupuncture with a w ide range of indications in combating aging[7].For aging-related cognitive declines or even cognitive diseases, acupuncture was reported to show prom ising clinical benefits in previous trials[8-10].However, the anti-aging mechanism is still indistinct, which arouses our interest in investigating the underlying mechanism of acupuncture in m itigating cognitive deficits via animal experiment.Previous studies revealed that the activation of nucleotide- binding oligomerization domain(NOD)-like receptor protein 3 (NLRP3) inflammasome played a critical role in long-term cognitive impairment[11-12].Furthermore,suppression of the activation of NLRP3 inflammasomes/ NLRP3-ASC-Caspase-1 signaling pathway was reported to be one of the prom ising neuro-protective strategies in ameliorating cognitive dysfunction[13-14]. However, if this signaling pathway is also involved in the mechanism of acupuncture in improving aging-related cognitive declines or reversing cognitive dysfunction is still ambiguous. This is the original intention of our research as well.

As a mature and well-established rodent model of brain aging in various anti-aging studies[15-16],D-galactose (D-gal)-induced cognitive deficit rat model was introduced to our experiment. It has been proven that rodents would experience learning and memory impairments after undergoing 4-8 weeks successive adm inistration of D-gal at 50-500 mg/(kg·bw)][17-18].Inflammatory response and oxidative stress induced by D-gal were reported to contribute to memory and synaptic dysfunction[19-20].Therefore,D-gal-induced neurochem ical and behavioral changes could mim ic multiple features of the natural process of brain aging[20-22].

In our experiment,after receiving electro- acupuncture (EA) for different terms, the model rats'behaviors were evaluated using Morris water maze(MWM) test, novel object recognition (NOR) test and step-down passive avoidance (SDPA) test. In addition,the expressions of NLRP3 inflammasome-associated proteins including NLRP3,ASC,and Caspase-1 were determined via Western blot (WB).

1 Materials and Methods

1.1 Animals and grouping

A total of 46 male aged Sprague-Daw ley (SD)rats[clean-grade,specific pathogen free,weighing(180±20)g]were provided by Shanghai Slake Laboratory Animal Co.,Ltd.,China[License Number:SYXK (Shanghai) 2017-0008] and random ly divided into 4 groups: a control group (n=10), a model group (n=12),an EA-7 d group (n=12) and an EA-21 d group (n=12). All rats were housed separately in temperature- and hum idity-controlled facilities [(23±2)℃,(50±5)%humidity] with a 12 h/12 h light/dark cycle, and free access to food and sterile water. All the rats were given 7 d for acclimatization before the commencement of experiment.

1.2 Main reagents and instruments

1.2.1 Main reagents

D-gal injection(Batch No.:F20170311,Chem ical Reagents Shanghai Co.,Ltd.,China National Pharmaceutical Group Corporation,China);NLRP3(Batch No.:ab4207)and Caspase-1(Batch No.:ab22731), [Abcam Trading (Shanghai) Co., Ltd., China];ASC [Batch No.: sc-514414, Santa Cruz Biotechnology (Shanghai) Co., Ltd., China]; β-actin (Batch No.: 4970L,Shanghai Rebiosci Biotechnology Co., Ltd., China).

1.2.2 Main instruments

Standard sterilized disposable needles (0.25 mm in diameter and 25 mm in length, stainless steels, Jiajian Medical Instrument Co., Ltd., China); G6805-2 electrical stimulator (Shanghai Huayi Medicinal Instruments Co.,Ltd., China); EthoVision? XT 9.0 video tracking system(version 9.0,Noldus,Netherlands);XR-XX117 NOR experimental video analysis system and XR-3TB SuperSdt SDPA video analysis system(Shanghai Softmaze Information Technology Co.,Ltd.,China);P0018 chemilum inescence analyzer (Beyotime Biotechnology Co.,Ltd.,China);Super RX films [FUJIFILM (China) Investment Co., Ltd., China].

1.3 Modeling

The model group, EA-7 d group and EA-21 d group received intraperitoneal injection of D-gal at 100 mg/(kg·bw)per day for 42 d to establish aging models.The control group received intraperitoneal injection of normal saline at the same dose.

1.4 Intervention methods

Each rat was restrained in a cylindrical plastic restrainer which only allowed the rat's hind legs to extend out. Standard sterilized disposable stainless steel needles of 0.25 mm in diameter and 25 mm in length were inserted into Dazhui(GV 14),Baihui(GV 20),Shenshu(BL 23)and Zusanli(ST 36)as deep as approximately 3-5 mm.The points were located according to thePractical Animal Acupuncture Manual[23]andExperimental Acupuncture Science[24].The locations of the points were listed in Table 1. The handles of the needles were attached to electrodes of the electrical stimulator.Dazhui(GV 14)and Baihui(GV 20) were connected to a pair of electrodes, and Shenshu (BL 23) and Zusanli (ST 36) on the same side were connected to a pair of electrodes for a total of three pairs. The EA parameters were set as follows: a sparse-dense wave at 2 Hz/15 Hz and 2 mA (causing slight vibration of muscles around the points). The EA intervention was offered once a day,20 min each session.

Table 1.Locations of each acupoint selected in this study

1.4.1 EA-21 d group

EA intervention in this group started from the 22nd day of modeling (intraperitoneal injection of D-gal), and finished on the 42nd day (the same day that the modeling process ended), for 21 successive days.

1.4.2 EA-7 d group

EA intervention in this group started from the 36th day of modeling, and finished on the 42nd day as well,for 7 successive days.

1.4.3 Control group and model group

Rats in both control group and model group were allowed free access to food and sterile water.Meanwhile, they were grasped and restrained in the same time as that in the EA group but did not receive any other stimulation or intervention.

1.5 Observations

1.5.1 Behavioral tests

The behavioral evaluation was determ ined by MWM,NOR and SDPA tests and commenced at the end of the 42-day modeling. The testing environment was required to be quiet w ith the room temperature controlled at 20-23℃,and the light was dim and remained constant.

MWM test: The water maze consists of a circular pool(130 cm×50 cm) made of black plastic and filled with water (25.0±0.5)℃. The maze was divided into four quadrants:E,S,W,and N quadrants.An invisible platform (10 cm×10 cm) was placed 2 cm below the water surface in the E quadrant (target quadrant). The rats were expected to find the hidden platform w ithin 60 s and allowed to have a rest on the platform for 10 s after each test. If rats failed to reach the platform w ithin the allowed time,they were guided towards the platform and left there for 10 s.All rats received repeated training for 5 consecutive days but would be removed if failed to find the platform on the 5th training day.

The 5 training days began after successful modeling,and then the place navigation test (PNT) was performed on the 6th day and lasted for 4 d. During the test, the platform was set at any of the four quadrants, and rats were placed at the farthest quadrant from the platform.The time spent to find the hidden platform was recorded as the escape latency.The escape latency would be recorded as 60 s if a rat failed to find the hidden platform. The PNT was followed by the spatial probe test (SPT). In the SPT without platform, the times of crossing the target quadrant where the hidden platform had been set (it was also called‘platform crossing numbers'), as well as target quadrant residence time(it was also called‘time spent in the target quadrant') were recorded within 60 s.

All the rats' movements were recorded by a video tracking apparatus mounted on the ceiling, and digital images were analyzed by the EthoVision video tracking system.

NOR test: The NOR test was completed over 3 d,habituation day, training day, and testing day. The NOR consists of a test box (80 cm×80 cm×60 cm) and three toys (A, B and C) made of sim ilar size but in different shapes, colors and textures.On the habituation day,after stroked,rats could explore and adapt to the environment in the box for 5 m in per day for 3 consecutive days. On the training day, toys A and B were put into the box. The total time of rats' exploring the toys (the time rats looked at, sniffed, or touched the toys within about 5 cm around the toys) w ithin 5 m in were recorded. On the testing day (24 h after the end of training day), toy A was replaced by toy C, and the time of rats exploring toys B and C w ithin 5 m in was recorded.At the end of each test, the test box and toys were w iped w ith 75% alcohol to elim inate the smell left by the previous rat. The NOR ratio [= Exploration time on toy C ÷ Exploration time on (toy B + toy C)] was taken as a parameter to evaluate the rats' cognitive ability. All the rats'movement tracks and performances were collected and analyzed by the NOR experimental video analysis system.

SDPA test:The rat's memory function was determ ined by the SDPA test using the SuperSdt SDPA video analysis system. The training apparatus of SDPA test consists of a plastic box (80 cm×40 cm×20 cm) and the floor of which was made of 0.1 centimeter-caliber stainless steel bars parallelly spaced 0.5 cm apart. An elevated rubber platform(12.5 cm in diameter and 4.5 cm in height) was placed by the left wall of the training box apparatus.Rats were first exposed to a 5-m inute learning course,during which they were perm itted to move freely through the chamber,and were then placed on the platform. If the rat stepped down from the platform (‘error signal'), they would be punished by an alternating current (AC) electric shock (36 V). After the shock, the rat would reflexively jump to the platform (safe area). When the rat stepped down from the platform again,it would experience the electric shock again. The numbers of ‘errors' (times that the rat received the electric shock) during 3 min were recorded to evaluate the ‘passive learning ability' of the rats.

After the test was completed, the rats were returned to the cages. Twenty-four hours later, the rats were placed on the platform again, and the time that the rat took to step down from the platform for the first time(step-down latency)w ithin 3 m in was recorded and taken as the measure of rat' memory retention.

1.5.2 Biomarkers test

After all the behavioral tests were completed, the rats were sacrificed and the expressions of the hippocampal target proteins NLRP3, ASC, and Caspase-1 were detected by WB.

Protein sample preparation: Rat's brain tissues sliced at 400 μm were incubated in the oxygenated artificial cerebral spinal fluid (ACSF) for 1 h. The hippocampus tissues were separated and transferred quickly to the frozen ACSF containing BS3(2 mmol).After centrifugation,the supernatant was discarded.The hippocampus tissues were cut into pieces on ice (4 ℃)and washed twice w ith PBS, then centrifuged again, and PBS was discarded. After adding 0.5-1.0 m L of radio immunoprecipitation assay(RIPA)lysis buffer,the samples were thoroughly homogenized by an electric homogenizer until 95% of the cells were broken. The samples were then placed in ice bath for 10 min and shaken on a vortex m ixer for 30 s every 5 min. After centrifugation at 12 000 g for 10 m in at 4 ℃,the supernatant was transferred to a new centrifuge tube to obtain the total protein product. Its concentration was determ ined by bicinchoninic acid (BCA)method.Finally, the samples were added w ith loading buffer,boiled for 10 min to fully denature the protein, and then stored in fridge (-80℃) for later use.

WB:Extraction of total protein;preparation of sodium dodecyl sulfate-polyacrylam ide gel electrophoresis(SDS-PAGE);loading samples (sample load of 100 μg); electrophoresis, 100 V, 1 h; placed the polyvinylidene fluoride(PVDF)membrane in the transfer tank w ith a constant current of 250 mA for 120 m in;the transfused PVDF membrane was immersed in 5% skim m ilk-Tris-buffered saline w ith Tween (TBS-T) solution for 1 h at room temperature;primary antibody incubation(NLRP3,1:1 000;ASC,1:500; Caspase-1, 1:1 000; β-actin: 1:3 000) at 4 ℃ for 12 h, and the membrane was then rinsed for 3 times by TBS-T for 5 m in each time;secondary antibody incubation at room temperature for 1 h,and the membrane was then rinsed for 3 times by TBS-T for 5 min each time; developed by chem ilum inescence for 5 min, and the films were then rinsed and air-dried after fixed for 10 m in; the images obtained by scanning the films were analyzed by Quantity One analysis software to obtain the gray value of the target brand,which was then compared w ith that of the β-actin to obtain the relative grey value of the target brand.

1.6 Statistical methods

IBM SPSS version 23.0 statistical software was used for statistical description and inference after the original data were inputted via Excel 2010.Data in normal distribution were expressed as mean±standard deviation(±s).The inter-group comparisons were analyzed by one-way ANOVA,followed by multiple comparisons with least significant difference(LSD)except that the data of escape latency in the MWM test were analyzed by ANOVA for repeated measurement.The significance level was set at 0.05.

2 Results

2.1 Behavioral tests

2.1.1 Comparison of the MWM test results

On PNT day 1, there was no significant difference in the escape latency among the control group,EA-7 d group and EA-21 d group (P＞0.05), and all of them were significantly shorter than that of the model group(P＜0.05). On day 2 and day 3, there was no significant difference in the escape latency between the control group and EA-21 d group(P＞0.05), and their escape latency were significantly shorter than that of the EA-7 d group and model group (P＜0.05); the escape latency of the model group was longer than that of the EA-7 d group(P＜0.05).On day 4,there was no significant difference in the escape latency between the control group and EA-21 d group(P＞0.05),and the escape latency were significantly shorter in these two groups than in the EA-7 d group and model group(P＜0.05); no significant difference was found between the EA-7 d group and model group comparing the escape latency (P＞0.05). The escape latency generally showed a downward trend with the time in the four groups (Figure 1).

In SPT, there was no significant difference comparing the time spent in the target quadrant between the control group and EA-21 d group (P＞0.05), and both of them were significantly shorter than those of the EA-7 d group and model group (P＜0.05); the time spent in the target quadrant was significantly shorter in the model group than in the EA-7 d group (P＜0.05), (Figure 2).

Figure 1. Com parison of the escape latency in PNT

Figure 2. Comparison of the time spent in the target quadrant

There were no significant differences in the average sw imm ing speed among the four groups (P＞0.05),(Figure 3). There was no significant difference in the platform crossing numbers among the control group,EA-7 d group and EA-21 d group (P＞0.05), and all of them were significantly higher than that of the model group (P＜0.05), (Figure 4).

2.1.2 Comparison of the NOR test results

There was no significant difference in the NOR ratio between the EA-7 d group and EA-21 d group (P＞0.05).The NOR ratio was significantly higher in these two EA groups than in the model group(P＜0.05),but lower than in the control group (P＜0.05), (Figure 5).

Figure 3. Comparison of the average sw imm ing speed

Figure 4. Com parison of the platform crossing numbers in SPT

Figure 5. Comparison of the NOR ratio

2.1.3 Comparison of the SDPA test results

There was no significant difference in the electric shock numbers among the control group, EA-7 d group and EA-21 d group(P＞0.05), and the numbers were higher in these three groups than in the model group(P＜0.05), (Figure 6).

There were significant differences among the four groups comparing the step-down latency (P＜0.05), and the time from long to short was the control group,EA-21 d group, EA-7 d group, and model group in order (P＜0.05), (Figure 7).

Figure 6.Comparison of the electric shock numbers

Figure 7. Comparison of the step-down latency

2.2 Comparison of the expressions of biomarkers

There was no significant difference in the expression of NLRP3 between the control group and EA-21 d group(P＞0.05). Compared w ith the control group and EA-21 d group, the expression of NLRP3 increased significantly in the model group and EA-7 d group (P＜0.05), and the expression in the model group was even higher (P＜0.05). There was no significant difference comparing the ASC and Caspase-1 expressions among the control group,EA-7 d group and EA-21 d group(P＞0.05).Compared w ith these three groups,the ASC and Caspase-1 expressions showed a significant increase in the model group(P＜0.05).Please see Figure 8 and Figure 9 for details.

Figure 8. Comparisons of the relative grey values of NLRP3, ASC and Caspase-1 in hippocampus

Figure 9. WB protein expressions of NLRP3, ASC and Caspase-1 in rat's hippocampus

3 Discussion

Aging is an intricate but inevitable natural process that is associated w ith loss of functional capacities in several body systems[1-2,25].Is pathology-free aging possible? This must be the common vision of mankind[26]while the reality is that aging-induced decline in multiple body functions leads to the occurrence or development of a variety of diseases,most of which are aging-related cognitive diseases[3-4,27],particularly neurodegenerative diseases[28].Although the detailed pathogenesis of cognitive dysfunction is unclear,emerging evidences have shown that the activation of NLRP3 inflammasome should be involved in the development of many neuro-degenerative diseases[11-12,29-30].NLRP3 inflammasome thereby has been speculated as a potential target for treating or preventing cognitive decline in the neuroinflammationassociated diseases or even for slowing down the cognitive decline in the natural aging process[29].

Can acupuncture improve cognitive dysfunction caused by brain aging via acting on NLRP3 inflammasome pathway (NLRP3-ASC-Caspase-1 signaling pathway)? This has aroused our great interest.An aging rat model w ith cognitive dysfunction induced by consecutive intraperitoneal injection of D-gal was introduced in our study[15-16]. Meanwhile, MWM, NOR and SDPA tests were used to assess the behavioral changes,while WB was used to determine the expressions of inflammasome-associated proteins of rats undergoing different interventions.

According to the results, following the 42 successive days of adm inistration of D-gal,compared w ith the control group,rats in the model group showed poor performance in behavioral tests,including longer escape latency,shorter time spent in the target quadrant and lower platform crossing numbers in MWM test, lower NOR ratio in NOR test, and higher electric shock numbers and shorter step-down latency in SDPA test. These findings suggested that the model was successful,that is,the rats showed significant cognitive impairments along w ith D-gal-induced brain aging. With the change of behaviors, the expressions of NLRP3, ASC, and Caspase-1 in rat's hippocampus were significantly increased,indicating that the cognitive dysfunction of rats m ight be associated w ith the activation of NLRP3 inflammasome pathway.Our results are consistent w ith the conclusions of previous research, that is, NLRP3 inflammasome plays a key role in the development of inflammatory responses in the central nervous system (CNS)[29]and w idely involved in several neurological disorders[29-31].Why does aging lead to the activation of NLRP3 inflammasome? Why and how does activated NLRP3 inflammasome damage cognitive function? The current study did not answer the above questions,but previous reports m ight provide valuable references as follows:NLRP3 inflammasome is abundantly expressed in the CNS during aging[31-33];activated NLRP3 inflammasome induces the activation of Caspase-1, which mediates the production of interleukin(IL)-18 and IL-1βproinflammatory cytokines and the initiation of a rapid form of cell death termed pyroptosis[34-35].IL-1β and IL-1b, in turn, initiate multiple signaling pathways and drive inflammatory responses, which w ill finally lead to neuronal injury or even apoptosis[36-38].

In summary,NLRP3 inflammasome may be a potential therapeutic target for m itigating the aginginduced cognitive impairments,which has also been confirmed in the current research.For instance,no significant difference was observed between the EA-21 d group and control group in MWM, suggesting that the damages of spatial learning and memory ability in rats resulted from the modeling process were improved after EA intervention. It should be noted that, in the navigation test, although the EA-7 d group performed similarly to the control group and the EA-21 d group on the 1st day, a gradual decline in the performance was found from the 2nd day to the 4th day, indicating that 7-day EA intervention had lim itations in improving spatial learning and memory ability. Sim ilar conclusion could be drawn from the result of ‘time spent in the target quadrant' in the spatial probe test. However, the sw imming speed did not seem to be highly correlated w ith the cognitive impairments. The episodic memory capability of rats was reflected by the results of the NOR test: rats receiving EA (no matter 7 d or 21 d) had better episodic memory capability than the model rats but were still worse than the rats in the control group.These findings revealed that EA could only partially improve the aging-induced episodic memory impairments,and the efficacy seemed to be weakly correlated w ith the length of intervention. The passive learning ability and memory retention ability were reflected by the results of the electric shock numbers and step-down latency, respectively, in the SDPA test.According to our results, EA can significantly m itigate the damages of passive learning ability, and the efficacy was weakly correlated with the length of intervention.However, aging-induced damages of memory retention ability can only be partially improved. Furthermore, the shorter the EA treatment course, the worse the effects.

Accompanied by the improved performance in the behavioral tests, notable decline in the expressions of NLRP3,ASC,and Caspase-1 could not be neglected,suggesting that EA-induced attenuation of cognitive dysfunction might be achieved by inhibiting the activated NLRP3 inflammasome.

NLRP3 inflammasome may be involved in the development of cognitive decline in aging rats though the detailed mechanism is indistinct currently concerning how aging triggers the NLRP3 inflammasome pathway and how the activated NLRP3 inflammasome induces the cognitive dysfunction of rats.Twenty-one consecutive days of EA intervention can improve the learning and memory ability in aging rats,which might be achieved by suppressing the activation of NLRP3 inflammasome(down-regulating the expressions of NLRP3 inflammasome-associated proteins in hippocampus).In addition,7 consecutive days of EA intervention can partially alleviate the cognitive impairments in aging rats though the effect is limited.

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Received:20December 2019/Accepted:22 January 2020