Berberine Ameliorates Lipopolysaccharide-Induced Cognitive Impairment Through SIRT1/NRF2/NF-κB Signaling Pathway in C57BL/6J Mice

Abstract

The inflammatory response is the stress reactions to infection or injury so as to help the body return to normal as soon as possible. In central nervous system, the overactivated immune system causes irreversible damage to neurons and synapses, which results in cognitive impairment. Berberine, an isoquinoline alkaloid extracted from Coptidis Rhizoma, plays a powerful role in anti-inflammation. It has been reported that berberine significantly improved the decline of cognitive ability. Therefore, we carried out this work to find out the specific mechanism. We tested behaviorally that berberine administration did improve lipopolysaccharide (LPS)-induced cognitive impairment in C57BL/6J mice. We found that berberine reduced neuronal damage in the hippocampus by Nissl staining, and verified by western blot and immunofluorescence that berberine improved LPS-induced cognitive impairment through the SIRT1/nuclear factor E2-related factor 2 (NRF2)/nuclear factor-kappaB (NF-κB) signaling pathway. The results showed that berberine plays an anti-inflammatory and antioxidant role by targeting SIRT1/NRF2/NF-κB signaling pathway so as to reduce the cognitive impairment and neuronal damage caused by LPS in C57BL/6J mice. Berberine preprotection increased the expression of heme oxygenase-1 (HO-1) after activating NRF2 and inhibited the activation of NF-κB and the release of inducible NO synthase, which may be related to berberine activating SIRT1. However, the effect of reducing inflammatory response was inhibited after using SIRT1 inhibitor EX527 in vitro. This research explains the significance of anti-inflammatory in the treatment of cognitive impairment from different angles.

Introduction

Inflammation is the response of body to infection or injury, which can help the body return to normal as soon as possible.¹ When the brain is damaged, brain cells will stimulate the immune cells of the body in some way. This study has been successfully validated in mice model of traumatic brain injury, stroke, and infectious diseases.^2

–5 Although the purpose of the immune system is to protect the body from damage, in some cases, the activation of the immune system will have a negative impact. The over activated immune system causes irreversible damage to neurons and synapses. Researches show that inflammation is the basis of many diseases and symptoms, including neurodegenerative diseases such as Alzheimer's disease,⁶ Parkinson's disease,⁷ amyotrophic lateral sclerosis,⁸ multiple sclerosis,⁹ mental diseases such as obsessive-compulsive disorder,¹⁰ anxiety disorder, depression,¹¹ and brain parenchymal injury diseases such as traumatic brain injury,² intracerebral hemorrhage,¹² and cerebral infarction.¹³

Furthermore, increasing evidences show the association between inflammation and cognitive impairment.¹⁴ A kind of diffuse brain dysfunction that occurs secondary to infection in the body without overt CNS infection is called sepsis-associated encephalopathy (SAE). According to the report by Gofton, up to 70% of patients with severe systemic infection encountered SAE.¹⁵ New report claims that approximately half of the patients with sepsis develop SAE in the intensive care unit, and some survivors present with sustained cognitive impairments for several years after initial sepsis onset.¹⁶ How does acute and chronic inflammation affect the brain? How do brain damaged signals activate the inflammatory responses? It is a potential target for the treatment of neurodegenerative diseases by reducing the harmful effects of inflammation and improving its protective effects.

Neuroinflammation, or more specifically, is the activation of neuroimmune cells, putting microglia and astrocytes into a preinflammatory state.¹⁷ Therefore, neuroimmune activation is often used to simulate the role of neuroinflammation. In the brain, microglia cells respond to the inflammation first. When being stimulated, microglia show morphological differences and rapidly differentiate into M1 and M2 cells. The M1 cells mainly play a role in recognition chemotaxis and phagocytosis. However, excessive activation of microglia can release a large number of proinflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, inducible NO synthase (iNOS), IL-23, CXCL10, and so on, leading to the occurrence of inflammatory storms.¹⁸

Lipopolysaccharide (LPS) is a major component of the cell wall of gram-negative bacteria, which is widely used to study the interference of inflammatory pathways. In LPS-stimulated macrophages, the expression of inflammatory cascade effector enzymes and cytokines are recognized to be mediated by toll-like receptor 4 (TLR4) and nuclear factor-kappaB (NF-κB).¹⁹ Nitric oxide (NO) and prostaglandin E2 (PGE2) is representative of the inflammation medium, while TNF-α, interleukins (IL1 β, IL6, IL12), and other cytokines promote inflammatory responses. Their inadequate activations or abnormal upregulations are common in most inflammatory diseases. iNOS and cyclooxygenase2 (COX-2) are involved in the production of NO and PGE2 and their activities are positively correlated with the expression of proinflammatory cytokines.^20,21 At the same time, reactive oxygen species (ROS) and its related species are important signal molecules necessary for normal physiological functions of the body and that are closely related to host defense responses.

However, oxidative stress characterized by excessive ROS production contributes to inflammatory responses and disease progression. LPS induces and accelerates inflammatory cascade and oxidative stress, also increases the production of ROS. Increasing evidences have shown a strong association between overproduction of inflammatory factors and accumulation of ROS.^22
–24 Currently, nonsteroidal anti-inflammatory drugs are widely used to relieve inflammatory symptoms, but various side effects have been reported after long-term usage.²⁵ Therefore, it is urgent to develop reliable and effective drugs to prevent and treat diseases mediated by inflammation and oxidative stress.

Berberine, an isoquinoline alkaloid extracted from Coptidis Rhizoma, has broad antibacterial effect on many gram-positive and gram-negative bacteria in vitro. Our previous study showed that berberine ameliorated diabetic encephalopathy by activating SIRT1.²⁶ In the meantime, the downstream events of SIRT1 activation remain unknown. However, previous study reported that berberine inhibits inflammation by activating nuclear factor E2-related factor 2 (NRF2).²⁷ In this study, we specifically explored whether berberine ameliorates LPS-induced cognitive impairment in C57BL/6J mice via SIRT1/NRF2/NF-κB signaling pathway.

Materials and Methods

Animal and drug treatment

All in vivo experiments were approved by the experimental animal Ethics Committee of Animal Experiment Center, Guangzhou University of Chinese Medicine. Eight to 10 weeks old male C57BL/6J mice weighing about 20–25 g were acquired from Guangdong Medical Laboratory Animal Center (Guangdong, China). The mice were exposed to 12-hour Light-Dark Cycle for a week before the experiment. Then they were randomly divided into five groups. Control group and model group were given the equal volume of normal saline by gavage. BBR-L group was intragastric with low-dose berberine (50 mg/kg), while BBR-H group was with high dose (100 mg/kg). Ctrl+BBR-H group was also administered with high dose.

On day 4 of intragastric administration, at the same time, all mice were intraperitoneally injected with LPS (1 mg/kg) (Solarbio, Beijing, China) or saline for a week. After completing the behavioral tests, all mice were intraperitoneally injected with 4% chloral hydrate and then dislocated the cervical after deep anesthesia.

Cell culture

BV2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in Dulbecco's modified Eagle's medium (Biological Industries, Israel), supplemented with 10% fetal bovine serum (Biological Industries) at 37°C under 5% CO2. LPS at 1 μg/mL was used to treat BV2 cells for 6 hours. Cells were treated with SIRT1 inhibitor EX527 (Abcam; ab141506) at 10 μmol/L for 18 hours, then treated with or without berberine at 10 μmol/L for 2 hours before stimulation with LPS at 1 μg/mL.

Behavioral tests

All the behavioral tests were performed at the end of LPS administrated, including the open-field test (OFT) and novel object recognition test (NOR). Behavioral performances were recorded using a video camera. All the tests were recorded in an animal behavior analysis system (Shanghai Jiliang Software Technology Co., Ltd., China). OFT was performed on day 11 to measure locomotor activity. The locomotor activity of the mice in an open-field area (40 × 40 × 40 cm) was recorded for 5 minutes. NOR was carried out as previous.²⁸ In brief, the NOR consisted of three different sessions: a habituation session, a training session, and a retention session. A mouse was habituated to a box (20 × 40 × 40 cm) by allowing it to explore the box without objects for 3 minutes for 3 days (habituation session).

Twenty-four hours after the last habituation session, the mouse underwent a 5 minutes training session of exposure to two identical objects in an open-field box. The time spent exploring each object was recorded by a video camera. After the training session, the mouse was returned to its home cage. After an interval of 24 hours, the mouse was returned to the same box containing two objects, one identical to the familiar object, but previously unused and one novel object. The mouse was allowed to explore for 5 minutes retention session, during which the amount of time exploring each object was recorded.

Throughout the experiments, the objects used were matched in terms of their physical complexity and emotional neutrality. A preference index, that is, the ratio of the amount of time spent exploring any one of the two objects (training session) or the novel object (retention session) to the total amount of time spent exploring both objects, was used to measure cognitive function. Between trials, the compartments were cleaned with 75% ethanol to remove odor cues.

Nissl staining

The whole brain tissue was placed in 4% paraformaldehyde for fixation. The fixed brain tissue was dehydrated and transparent through the gradient of alcohol and xylene. Afterward, the brain was embedded in paraffin and sliced into 4 μm slices by a microtome. The slices of brain were stained by Nissl staining kit (Solarbio). These images were obtained under a microscope (Nikon, Tokyo, Japan) to observe morphological changes in hippocampus and cortex.

Western blot

Protein was extracted from tissue/cells using radio immunoprecipitation assay lysis buffer containing protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). After measuring protein concentration using a Bicinchoninic Acid kit (Beyotime), the proteins were subjected to 10% sodium dodecyl-sulfate-polyacrylamide gel for separation and then transferred onto a polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany).

Blocked with 5% nonfat milk for 2 hours at room temperature, the membranes were incubated with rabbit anti-iNOS antibody (CST; no. 13120s), rabbit anti-NF-κB p65 antibody (Abmart; T55034), rabbit anti-Phospho-NF-κB p65 antibody (Abmart; TP56372), rabbit anti-SIRT1 antibody (CST; no. 9475s), rabbit anti-Phospho-NRF2 antibody (Abmart; TD7519), rabbit anti-NRF2 antibody (CST; no. 12721s), rabbit anti-heme oxygenase-1 (HO-1) antibody (CST; no. 86806s), rabbit anti-NQO1 antibody (Abcam; no. ab80588), rabbit anti-β-Actin antibody (CST; no. 4970s), and rabbit anti-GAPDH antibody (Affinity; AF7021) at 4°C overnight, after which anti-rabbit IgG (H+L) secondary antibody (CST; no. 14708s) was incubated for 1 hour at room temperature. Immunoblots were visualized using enhanced chemiluminescence (Thermo Scientific).

Cell Counting Kit-8

BV2 cells were inoculated in 96-well plates (5 × 103 cells/well) and cultured at 37°C overnight. After LPS treatment, each well was added with 10 μL of Cell Counting Kit-8 (CCK-8) agent (Dojingdo Molecular Technologies, Inc.) and the cells were incubated with mixture for another 2 hours. Eventually, a microplate reader (Bio-Rad Laboratories, France) was used to determine the absorbance at 450 nm.

Immunofluorescence: After washing with phosphate-buffered saline (PBS) twice, the cells were fixed with 4% para-formaldehyde upon for 15 minutes at room temperature, then washed with PBS twice. Cells were permeabilized with PBS containing 0.5% Triton-X-100 at 37°C for 30 minutes then blocked in 5% bovine serum albumin for 2 hours at room temperature. The primary rabbit anti-iNOS antibody (CST; no. 13120s), rabbit anti-Phospho-NRF2 antibody (Abmart; TD7519), and rabbit anti-SIRT1 antibody (CST; no. 9475s) diluted 1:1000,1:200, and 1:500, respectively, in blocking solution was then added and incubated with cells at 4°C overnight. After three-time washes in PBS, the cells were incubated with Alexa Fluor 555 conjugated anti-rabbit secondary antibody (CST; no. 8953) for 2 hours at room temperature. Washed in dark four times with PBS and sealed with Antifade Mounting Medium with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (Beyotime; P0131). Images were captured immediately using a microscope (Nikon).

Statistical analysis

All results are represented as mean ± standard error of mean. Comparisons were made by one-way analysis of variance followed by Tukey's test. Statistical analysis was performed by GraphPad Prism V.8.0.0. (San Diego, CA). Values of p < 0.05 were considered statistically significant. All analyses were performed with three independent experiments.

Results

Berberine alleviates cognitive impairment caused by LPS

In our study, LPS group mice reduced total exploring distance and spent less time in central region, which was significantly different from the control group. However, mice pretreated with berberine increased the exploring distance and spent more time in central area, especially the high-dose group (Fig. 1A–C). To verify this result, we carried out the new object recognition test. It turned out that recognition index (RI) in LPS group was significantly lower than that in control group, while high dose of berberine pretreated reversed the result (Fig. 1D, E). These results showed that LPS induced cognitive impairment in mice, while berberine improved LPS-induced cognitive impairment in mice.

FIG. 1.

Effect of berberine on cognitive impairment caused by LPS. (A) The OFT locomotor activity. (B, C) Compared with the control group, the total distance of exercise and the exploration time of central area decreased in the model group. Compared with model group, LPS+BBR-L group did not change significantly, while LPS+BBR-H group significantly improved exercise distance and exploration time. (D) The NOR locomotor activity. (E) Compared with the control group, the RI of model group decreased, and BBR-H group effectively increased the RI. There was no difference between control+BBR-H group and control group. Data are expressed as mean ± SEM (n = 7).*p < 0.05; ***p < 0.01. BBR-H, high dose of berberine; BBR-L, low dose of berberine; Ctrl, control group; LPS, lipopolysaccharide; NOR, novel object recognition test; OFT, open-field test; RI, recognition index; SEM, standard error of mean.

Berberine reduces the damage of Nissl bodies in hippocampus and cortex

To observe the structure of neurons, we stained the mouse brain slices after finishing the behavioral test. As the result showed, the cortical and hippocampal CA1 neurons in the model group were enlarged, vacuolated, and shriveled compared with the control group. Berberine modified the damage of neurons in a dose-dependent way (Fig. 2). This result suggested that LPS-induced cognitive impairment may be related to the damage of neurons and berberine improved LPS-induced cognitive impairment by protecting neurons from damage.

FIG. 2.

Effect of berberine on changes of hippocampus and cortex induced by LPS. Compared with the control group, the cortical and hippocampal CA1 neurons in the model group were enlarged, vacuolated, and shriveled. Berberine reversed these changes in a dose-dependent manner. All photos are taken in uniform size (scale bars, 50 μm).

Berberine inhibited LPS-induced cerebral neuroinflammation and activated the SIRT1/NRF2 pathway in western blot assay in C57BL/6J mice

As we discussed above, LPS is a powerful inflammation-inducing agent. To explore whether LPS induced neuroinflammation in mice, we took the brains for western blot detection. Not surprisingly, compared with the control group, the expression of iNOS, p-NFkB P65 significantly upregulated in LPS group, while berberine high-dose group effectively inhibited this upregulations (Fig. 3). Then, we detected the expression of SIRT1 and NRF2. Mice stimulated with LPS reduced the expression of SIRT1 and increased the expression of NRF2. Berberine did improved SIRT1 expression but had little effect on NRF2 expression.

FIG. 3.

Berberine inhibited LPS-induced cerebral neuroinflammation by activating the SIRT1/NRF2/NF-κB pathway in western blot assay in C57BL/6J mice. (A, D) Representative western blot images. (B, C, E–I) Summarized data. Compared with the control group, the expression of iNOS, p-NF-κB P65 significantly upregulated, and the expression of SIRT1, p-NRF2 together with its downstream HO-1 and NQO1 were downregulated in model group, while berberine high dose administration effectively inhibited the upregulation of iNOS and p-NF-κB P65. At the same time, these downregulated proteins aforementioned were markedly reversed. Moreover, LPS stimulation effectively increased the expression of NRF2, and berberine did not affect the expression of NRF2. Data are expressed as mean ± SEM (n = 3).*p < 0.05; ***p < 0.01. HO-1, heme oxygenase-1; NF-κB, nuclear factor-kappaB; NRF2, nuclear factor E2-related factor 2; iNOS, inducible NO synthase.

However, after berberine treatment, NRF2 downstream target genes HO1 and NQO1 were indeed upregulated compared with LPS group. Next, we assessed the phosphorylation of NRF2 and found that it was consistent with the trend of downstream genes expression (Fig. 4). A scientific hypothesis of the mechanism came out based on this experiment. Berberine inhibited LPS-induced neuroinflammation by upregulating SIRT1 and activating NRF2 to produce HO-1/NQO1. Thus berberine was able to improve the cognitive impairment of mice caused by LPS.

FIG. 4.

Optical microscopy of BV2 cells. Under optical microscope, BV2 in control group was round or oval, with or without short tentacles. Berberine treatment not morphologically affect BV2 cells; LPS enlarged the cell body, extended the branch length; berberine preprotection kept BV2 away from the morphological changes caused by LPS, while EX527 restrained the effect of berberine. All photos are taken in uniform size (scale bars, 50 μm).

Berberine activated SIRT1/NRF2/NF-κB signaling pathway to anti-inflammation in BV2 cells

To demonstrate the hypothesis, we tested and verified it on BV2 cells. First, we screened the safe range of berberine between 0, 5, 10, 20, and 40 μM by cell viability assay. It showed that berberine was toxic to BV2 cells at 20 μM and above (Fig. 5A). Second, according to the western blot assay results of iNOS, we chose berberine at the concentration of 10 μM among 2.5, 5, and 10 μM as the best dose for in vitro administration (Fig. 5B, C). Then we observed the morphology of different groups of cells under a light microscope. It showed that BV2 in control group was round or oval, with or without short tentacles. Berberine treatment did not morphologically affect BV2 cells, while LPS enlarged the cell body, extended the branch length.

FIG. 5.

Optimum dose selection of berberine for BV2 cells. (B) CCK8 assay was carried out on BV2 cells with concentration gradient of berberine5, 10, 20, and 40 μM. Berberine20 μM and above showed the toxicity to BV2 cells. Then berberine2.5, 5, and 10 μM were groped for the optimal concentration. (A, D, G) Representative western blot images. (C, E, F, H–L) Summarized data. Berberine10 μM was founded to be the best according to the expression of iNOS protein. Consistent with the results of animal tissue, berberine activated SIRT1/NRF2/NF-κB signaling pathway to anti-inflammation in BV2 cells (D, G). LPS administration decreased the expression of SIRT1, P-NRF2, and their downstream proteins HO-1 and NQO1, and increased the expression of iNOS and p-NF-κB P65 in BV2 cells. However, berberine10 μM preadministration reversed these changes, and the protective effect of berberine was inhibited by SIRT1 inhibitor EX527. Data are expressed as mean ± SEM (n = 3). *p < 0.05; ***p < 0.01. CCK8, Cell Counting Kit-8.

However, berberine preprotection kept BV2 away from the morphological changes caused by LPS, but EX527 restrained the effect of berberine (Fig. 4). Immunofluorescence staining showed that berberine reduced LPS—induced iNOS, while SIRT1 inhibitor EX527 inhibited this protective effect (Fig. 6). This demonstrated that berberine inhibited inflammation by targeting SIRT1. Later, we used WB assay to verify that berberine improved neuroinflammation by activating SIRT1/Nrf2/NF-κB signaling pathway (Fig. 5D, G). Finally, the key target proteins of this pathway SIRT1 and Phospho-Nrf2 were verified by immunofluorescence staining (Fig. 6).

FIG. 6.

The effect of berberine on iNOS, SIRT1, and p-NRF2 induced by LPS in BV2 cells monitored by immunofluorescence microscopy. LPS administration induced iNOS and diminished SIRT1 and p-NRF2 in BV2 cells, while berberine preprotection effectively reduced the expression of iNOS, normalized the expression of SIRT1 and p-NRF2. However, these effects of berberine were relieved when SIRT1 inhibitor EX527 was given in advance. All photos are taken in uniform size (scale bars, 50 μm).

Discussion

In the present study, we showed that berberine ameliorated LPS-induced behavior abnormalities in a mouse model of acute systemic inflammation, and decreased NF-κB activation in mice. Berberine administration improved the Nissl bodies' damage of CA1 and cortex in mice as evidenced by Nissl staining. What's more, berberine activated SIRT1 and NRF2 in mice by western blot. In vitro experiments confirmed these results and revealed the beneficial effect of berberine on SIRT1 and NRF2 activation in LPS challenged microglia.

Neuroinflammation (or inflammation of the brain), or the excessive activation of the innate immune cells in the brain microglia, is not only a result of disease progression but also an essential upstream mechanism for disease progression. According to a recent report, inappropriate immune responses and excessive neuroinflammation are the primary culprits of neuronal death in Alzheimer's Disease-related cognitive damage.²⁹ If neuroinflammation is taken as the metaphor of “forest fire,” then high levels of amyloid beta deposition and tau tangles are just fuses of the fire. When a fire has already broken out, controlling inflammation in time is the most important measure to put out fires, save more neurons so as to minimize losses.

Another study suggested that targeting neuroinflammation may benefit people in the early stages of Alzheimer's Disease and may also help reverse or at least slow down the accumulation of pathological tau proteins in the brain and stave off dementia.³⁰ In a nutshell, studies have shown that limiting inflammation would be beneficial for dementia treatment. Berberine is a natural isoquinoline alkaloid extracted from Coptidis Rhizoma that has a variety of pharmacological effects. Recently, increasing evidences show that berberine can improve cognitive impairment.³¹ In this study, cognitive impairment appeared in LPS-induced mice, oral administration of berberine significantly improved the learning and memory impairment in C57BL/6J mice.

We used the OFT and the new object recognition test to prove the successful build of LPS-induced cognitive impairment in mice and to find out the role of berberine in LPS-induced cognitive impairment. The OFT is used to analyze locomotion, anxiety and stereotypical behaviors such as grooming and rearing in rodents.²⁸ For mice, the test area normally consists of a 40 × 40 × 40 cm polyvinyl chloride box and a camera used to monitor movement into and around the central and peripheral areas of the box. Changes in locomotion can be indicative of altered neurological processes and may therefore reflect abnormal brain function. According to our OFT result, LPS group mice reduced total exploring distance and spent less time in central region, which showed the successfully build of the model.

Similarly, the following NOR result supported this viewpoint. The new object recognition test is based on the innate preference of the rodent to explore the novel object rather than the familiar one. Therefore, a rodent that remembers the familiar object will spend more time exploring the novel object,³² which make this test quite popular in experimental research on memory of brain diseases or aging studies. Here, the RI was used to quantify the cognitive impairment. Compared with the control group, the RI of the model group declined statistically significant. On this basis, berberine administration reversed these changes. Therefore, we suggested that berberine could ameliorate LPS-induced cognitive impairment in C57BL/6J mice. According to the results of Nissl staining, LPS might cause cognitive impairment in mice by damaging the brain CA1 and cortex Nissl bodies. However, the underlying mechanism remained unknown.

It has been widely reported that NRF2 was able to initiate antioxidant action and inhibit inflammation.³¹ It is known to all that, under normal condition, NRF2 was present and was degraded as NRF2-KEAP1-CUL3 complex in the cytoplasm in a cycle of about 20 minutes.³³ However, excessive ROS accumulation activates tyrosine kinases to dissociate NRF2/Keap1 complex.^34,35 The accumulation of NRF2 in the cytoplasm entered the nucleus and coordinated activation of cytoprotective gene expression. Nevertheless, the signal upstream of NRF2 is unclear and the exact form of activation of NRF2 is also controversial. In this study, we found that LPS did increase the expression of NRF2. However, it was the activation of phospho-NRF2 that played a protective role by increasing the downstream HO-1 and NQO1 expression.

SIRT1 is a member of the nicotinamide adenine dinucleotide (NAD⁺)-dependent deacylase family. It has been extensively studied in regulating homeostasis, improving organ function, enhancing physical endurance, resisting disease, and even reversing aging.^36,37 Many studies have reported that berberine was able to regulate metabolic disorder, anti-inflammatory, and repair damage and maintain homeostasis by increasing SIRT1 expression.^38
–40 In this study, LPS reduced the expression of SIRT1, while berberine activated SIRT1. The expression of SIRT1 was consistent with p-NRF2 rather than NRF2. In vitro BV2 cells experiments illustrated that SIRT1 is key target by using SIRT1 inhibitor EX527.

The protective effects of berberine on LPS-challenged microglia were inhibited after EX527 treatment. However, there is much more to be done. For example, EX527 should be used in vivo to validate the effect of SIRT1 inhibition on this pathway. A more convincing method would be to verify the results with SIRT1 knockout mice. For various reasons, further verification will be carried out by future studies.

Conclusions

In summary, in vivo and in vitro experiments demonstrated that berberine was able to improve cognitive impairment and reduce neuroinflammation caused by LPS in mice, which may be related to SIRT1/NRF2/NF-κB signaling pathway.

Footnotes

Authors' Contributions

Y.-B.C. and Q.W. designed the article, N.C. collected the data, L.-L.F. and Y.-H.Z. analyzed the data, X.-C.W. reviewed the article, and Y.-B.C. agreed to submit the article. All authors read and approved the final article.

Ethical Approval and Consent to Participate

The research protocols were approved by the Ethics Committee of Animal Experiment Center, Guangzhou University of Chinese Medicine.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by Key laboratory project of colleges and universities in Guangdong province (no. 2019KSYS005), National Natural Science Foundation of China (no. 82004246) and Science and Technology Program of Guangzhou (no.202201011714).

References

Leuti

, Fazio

, Fava

, et al. Bioactive lipids, inflammation and chronic diseases. Adv Drug Deliv Rev, 2020; 159:133–169; doi: 10.1016/j.addr.2020.06.028

Willis

, Macdonald

KPA

, Nguyen

, et al. Repopulating microglia promote brain repair in an IL-6-dependent manner. Cell, 2020; 180(5):833.e816–846.e816; doi: 10.1016/j.cell.2020.02.013

Bolte

, Lukens

. Neuroimmune cleanup crews in brain injury. Trends Immunol, 2021; 42(6):480–494; doi: 10.1016/j.it.2021.04.003

Shi

, Zou

, Jia

, et al. tPA mobilizes immune cells that exacerbate hemorrhagic transformation in stroke. Circ Res, 2021; 128(1):62–75; doi: 10.1161/circresaha.120.317596

Kodali

, Chen

, Liao

. Temporal unsnarling of brain's acute neuroinflammatory transcriptional profiles reveals panendothelitis as the earliest event preceding microgliosis. Mol Psychiatry, 2021; 26:3905–3919; doi: 10.1038/s41380-020-00955-5

Blanchard

, Victor

, Tsai

. Dissecting the complexities of Alzheimer disease with in vitro models of the human brain. Nat Rev Neurol, 2022; 18:25–39; doi: 10.1038/s41582-021-00578-6

Harms

, Ferreira

, Romero-Ramos

. Periphery and brain, innate and adaptive immunity in Parkinson's disease. Acta Neuropathol, 2021; 141(4):527–545; doi: 10.1007/s00401-021-02268-5

Stephenson

, Nutma

, Van Der Valk

, et al. Inflammation in CNS neurodegenerative diseases. Immunology, 2018; 154(2):204–219; doi: 10.1111/imm.12922

Wallin

, Culpepper

, Campbell

, et al. The prevalence of MS in the United States: A population-based estimate using health claims data. Neurology, 2019; 92(10):e1029–e1040; doi: 10.1212/wnl.0000000000007035

10.

Wang

, Chen

, Chiang

, et al. Systemic autoimmune diseases are associated with an increased risk of obsessive-compulsive disorder: A nationwide population-based cohort study. Soc Psychiatry Psychiatr Epidemiol, 2019; 54:507–516; doi: 10.1007/s00127-018-1622-y

11.

Du Preez

, Eum

, Eiben

, et al. Do different types of stress differentially alter behavioural and neurobiological outcomes associated with depression in rodent models? A systematic review. Front Neuroendocrinol, 2021; 61:100896; doi: 10.1016/j.yfrne.2020.100896

12.

Xue

, Yong

. Neuroinflammation in intracerebral haemorrhage: Immunotherapies with potential for translation. Lancet Neurol, 2020; 19(12):1023–1032; doi: 10.1016/s1474-4422(20)30364-1

13.

Raffaele

, Gelosa

, Bonfanti

, et al. Microglial vesicles improve post-stroke recovery by preventing immune cell senescence and favoring oligodendrogenesis. Mol Ther, 2021; 29(4):1439–1458; doi: 10.1016/j.ymthe.2020.12.009

14.

Savi

, De Oliveira

, De Medeiros

, et al. What animal models can tell us about long-term cognitive dysfunction following sepsis: A systematic review. Neurosci Biobehav Rev, 2021; 124:386–404; doi: 10.1016/j.neubiorev.2020.12.005

15.

Gofton

, Young

. Sepsis-associated encephalopathy. Nat Rev Neurol, 2012; 8:557–566; doi: 10.1038/nrneurol.2012.183

16.

Manabe

, Heneka

. Cerebral dysfunctions caused by sepsis during ageing. Nat Rev Immunol, 2022; 22:444–458; doi: 10.1038/s41577-021-00643-7

17.

Hickman

, Izzy

, Sen

, et al. Microglia in neurodegeneration. Nat Neurosci, 2018; 21(10):1359–1369; doi: 10.1038/s41593-018-0242-x

18.

Simões

JLB

, De Araújo

, Bagatini

. Anti-inflammatory therapy by cholinergic and purinergic modulation in multiple sclerosis associated with SARS-CoV-2 infection. Mol Neurobiol, 2021; 58(10):5090–5111; doi: 10.1007/s12035-021-02464-0

19.

Lauterbach

, Hanke

, Serefidou

, et al. Toll-like receptor signaling rewires macrophage metabolism and promotes histone acetylation via ATP-citrate lyase. Immunity, 2019; 51(6):997.e1017–1011.e1017; doi: 10.1016/j.immuni.2019.11.009

20.

Cinelli

, Do

, Miley

, et al. Inducible nitric oxide synthase: Regulation, structure, and inhibition. Med Res Rev, 2020; 40(1):158–189; doi: 10.1002/med.21599

21.

Xue

, Wang

, Lyu

, et al. PGE2/EP4 skeleton interoception activity reduces vertebral endplate porosity and spinal pain with low-dose celecoxib. Bone Res, 2021; 9(1):36; doi: 10.1038/s41413-021-00155-z

22.

Thatikonda

, Pooladanda

, Sigalapalli

, et al. Piperlongumine regulates epigenetic modulation and alleviates psoriasis-like skin inflammation via inhibition of hyperproliferation and inflammation. Cell Death Dis, 2020; 11:21; doi: 10.1038/s41419-019-2212-y

23.

Wang

, Pan

, Long

, et al. Endogenous hydrogen sulfide ameliorates NOX4 induced oxidative stress in LPS-stimulated macrophages and mice. Cell Physiol Biochem, 2018; 47(2):458–474; doi: 10.1159/000489980

24.

Peng

, Yang

, Tang

, et al. Therapeutic benefits of apocynin in mice with lipopolysaccharide/D-galactosamine-induced acute liver injury via suppression of the late stage pro-apoptotic AMPK/JNK pathway. Biomed Pharmacother, 2020; 125:110020; doi: 10.1016/j.biopha.2020.110020

25.

Palmer

, Clegg

. Salicylate toxicity. N Engl J Med, 2020; 382:2544–2555; doi:10.1056/NEJMra2010852

26.

, Wang

, Xu

, et al. Berberine improves diabetic encephalopathy through the SIRT1/ER stress pathway in db/db mice. Rejuvenation Res, 2018; 21(3):200–209; doi: 10.1089/rej.2017.1972

27.

, Wang

, Zhang

, et al. The crosstalk between Nrf2 and AMPK signal pathways is important for the anti-inflammatory effect of berberine in LPS-stimulated macrophages and endotoxin-shocked mice. Antioxid Redox Signal, 2014; 20(4):574–588; doi: 10.1089/ars.2012.5116

28.

Kraeuter

, Guest

, Sarnyai

. The open field test for measuring locomotor activity and anxiety-like behavior. Methods Mol Biol, 2019; 1916:99–103; doi: 10.1007/978-1-4939-8994-2_9

29.

Mcalpine

, Park

, Griciuc

, et al. Astrocytic interleukin-3 programs microglia and limits Alzheimer's disease. Nature, 2021; 595(7869):701–706; doi: 10.1038/s41586-021-03734-6

30.

Pascoal

, Benedet

, Ashton

, et al. Publisher correction: Microglial activation and tau propagate jointly across Braak stages. Nat Med, 2021; 27(11):2048–2049; doi: 10.1038/s41591-021-01568-3

31.

Chen

, Chen

, Liang

, et al. Berberine mitigates cognitive decline in an Alzheimer's Disease Mouse Model by targeting both tau hyperphosphorylation and autophagic clearance. Biomed Pharmacother, 2020; 121:109670; doi: 10.1016/j.biopha.2019.109670

32.

Leger

, Quiedeville

, Bouet

, et al. Object recognition test in mice. Nat Protoc, 2013; 8(12):2531–2537; doi: 10.1038/nprot.2013.155

33.

Zhou

, Gao

, Vong

, et al. Rhein regulates redox-mediated activation of NLRP3 inflammasomes in intestinal inflammation through macrophage-activated crosstalk. Br J Pharmacol, 2022; 179(9):1978–1997; doi: 10.1111/bph.15773

34.

Yarosz

, Chang

. The role of reactive oxygen species in regulating T cell-mediated immunity and disease. Immune Netw, 2018; 18(1):e14; doi: 10.4110/in.2018.18.e14

35.

Liu

, Pi

, Zhang

. Signal amplification in the KEAP1-NRF2-ARE antioxidant response pathway. Redox Biol, 2022; 54:102389; doi: 10.1016/j.redox.2022.102389

36.

Katsyuba

, Mottis

, Zietak

, et al. De novo NAD(+) synthesis enhances mitochondrial function and improves health. Nature, 2018; 563(7731):354–359; doi: 10.1038/s41586-018-0645-6

37.

Mitchell

, Bernier

, Aon

, et al. Nicotinamide improves aspects of healthspan, but not lifespan, in mice. Cell Metab, 2018; 27(3):667.e664–676.e664; doi: 10.1016/j.cmet.2018.02.001

38.

, Zhao

, Yang

, et al. Berberine potentiates insulin secretion and prevents β-cell dysfunction through the miR-204/SIRT1 signaling pathway. Front Pharmacol, 2021; 12:720866; doi: 10.3389/fphar.2021.720866

39.

Zhang

, Liu

, Xian

, et al. Berberine enhances survival and axonal regeneration of motoneurons following spinal root avulsion and re-implantation in rats. Free Radic Biol Med, 2019; 143:454–470; doi: 10.1016/j.freeradbiomed.2019.08.029

40.

Hassanein

EHM

, Kamel

, Ali

FEM

, et al. Berberine and/or zinc protect against methotrexate-induced intestinal damage: Role of GSK-3β/NRF2 and JAK1/STAT-3 signaling pathways. Life Sci, 2021; 281:119754; doi: 10.1016/j.lfs.2021.119754