Complementary and alternative medicine on cognitive defects and neuroinflammation after sepsis

Abstract

Sepsis-associated encephalopathy (SAE) is a common manifestation of sepsis, ranging from mild confusion and delirium to severe cognitive impairment and deep coma. SAE is associated with higher mortality and long-term outcomes, particularly substantial declines in cognitive function. The mechanisms of SAE probably include neuroinflammation that is mediated by systemic inflammation and ischemic lesions in the brain, a disrupted blood-brain barrier, oxidative stress, neurotransmitter dysfunction, and severe microglial activation. Increasing evidence suggests that complementary and alternative medicine, especially Traditional Chinese Medicine (TCM), is favorable in alleviating cognitive decline after sepsis. Here, we summarized the studies of traditional herbal remedies, TCM formulas and acupuncture therapy in animal models of neurological dysfunctions after sepsis in recent decades and reviewed their potential mechanisms.

1. INTRODUCTION

Sepsis has become a serious complication threatening human health, causing 11 million deaths worldwide every year.¹ Sepsis can lead to fatal organ dysfunction caused by dysregulation of the host response to infection.² Central nervous system (CNS) dysfunction often occurs in the acute and late stages of sepsis and is defined as sepsis-associated encephalopathy (SAE).³ SAE was found in up to 70% of the patients admitted to the intensive care unit and was associated with higher hospital mortality and poorer long-term neurological sequelae, especially cognitive decline. In SAE patients, neuroimaging and electrophysiological abnormalities can be detected in a wide range of brain regions, including the brainstem, cortex and hippocampus. The patients have clinical manifestations ranging from mild mental disorder and delirium to severe cognitive impairment and deep coma. The mechanism of SAE is complex. During sepsis, a disrupted blood-brain barrier (BBB) leads to cytokine and neutrophil infiltration, activates local astrocytes and microglia and amplifies neuroinflammation by releasing multiple inflammatory mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. Glial cell activation enhances the production of reactive oxygen species (ROS), which also lead to increased neuronal apoptosis.^4,5 In addition, the inflammatory reaction promotes changes in many neurotransmitter systems, such as brain-derived neurotrophic factor and serotonin, leading to an imbalance in neurotransmitter systems and increasing the incidence of brain injury.^6,⇓-8 To date, the treatment strategy for SAE is still limited. In recent years, accumulating evidence has shown that complementary and alternative medicine has potential therapeutic effects on SAE. In this review, we summarized the studies on complementary and alternative medicine, including traditional herbal remedies, Traditional Chinese Medicine (TCM) formulas and acupuncture therapy, and discussed the potential mechanisms in the treatment of SAE.

2. BEHAVIORAL CHANGES

Clinically, sepsis can lead to changes in cognitive, mood and motor function, including spatial cognitive impairment, anxiety, depression, irritability, anhedonia, and confusion.^9,⇓,⇓-12 In animal models of sepsis induced by cecum ligation and puncture (CLP) and lipopolysaccharide (LPS), memory and motor balance impairments were observed 24 h after surgery, which were detected using a novel object recognition test, T maze test, and rotarod and activity cage tests.¹³ At ten days after LPS- or CLP-induced sepsis, multiple behavioral dysfunctions were observed, including aversive memory, learning ability, depressive-like behavior, and exploratory and locomotor activities, which were assessed by the step-down inhibitory avoidance test, continuous multiple-trial inhibitory avoidance test, forced swimming test, elevated plus maze test, sweet consumption test, and open field test.^{14,⇓,⇓,⇓-18} Using the elevated plus maze, novel object recognition, and tail-suspension tasks (depressive-like behavior), one study showed cognitive impairment in rodents 28 d after LPS injection.¹⁹

Traditional herbal remedies, formulas and electroacupuncture have shown remarkable efficacy in treating cognitive dysfunction after sepsis. Using forced swimming, tail-suspension and sucrose preference tests, studies have shown that resveratrol,²⁰ curcumin²¹ and geniposide²² ameliorate LPS-induced increases in immobility time and LPS-induced reductions in sucrose preference. In the Morris water maze and Y maze tests, 6-gingerol significantly alleviated LPS-induced learning and memory impairments.²³ Mahuang Fuzi Xixin decoction (麻黄附子细辛汤) and Jiaotai Wan (交泰丸) alleviated LPS-induced depression-like behavior, as assessed by forced swimming, tail suspension, and sucrose preference tests, and improved the ability to explore the new environment in the open field test.^24,25 Qiang Xin 1 formula (强心1号) significantly alleviated CLP-induced memory impairment and anxiety-like behavior as assessed by novel object recognition testing, Morris water maze, elevated plus maze, and open field testing.²⁶ In addition, electroacupuncture alleviated learning and memory impairment in septic rats, as assessed by the Morris water maze test.^27,⇓-29

3. CYTOKINE RELEASE AND INFLAMMATORY SIGNALING PATHWAYS

Sepsis leads to a systemic inflammatory response mediated by the activation of the innate immune system. Pattern recognition receptors (PRRs) in immune cells, such as toll-like receptors, induce an intracellular signaling response and initiate a cytokine storm after detecting pathogens.^30,31 During this process, nuclear factor kappa-B (NF-κB) is triggered.³² Activation of NF-κB leads to the upregulation of cytokine gene expression, including IL-1, IL-18, IL-12, TNF-α, and type I interferons (IFNs).³³ These upregulated cytokines trigger inflammatory cascades that lead to the activation of other inflammatory factors and chemokines, including IL-6 and IL-8.^34,35 These peripheral inflammatory cytokines cross the BBB and enter the CNS and then activate the corresponding neural nuclei, ultimately leading to severe neurological damage and behavioral changes.^9,36,37 Maciel et al ³⁸ followed patients discharged from the intensive care unit for four years and showed that plasma IL-6 and IL-10 levels were associated with long-term functional and cognitive performance. IL-6 can stimulate the expression of COX-2 in brain glial cells to increase prostaglandin synthesis, especially prostaglandin 2, which activates the hypothalamic‒pituitary-adrenal axis (HPA axis), leading to behavioral changes in patients.³⁹

Therefore, anti-inflammatory agents are essential for the treatment of neuroinflammation and cognitive deficits after sepsis. Ginsenoside Rg3,⁴⁰ resveratrol,^20,41 curcumin,^21,42 6-gingerol,⁴³ isoginkgetin,⁴⁴ Bingpian (Borneolum Syntheticum),⁴⁵ quercetin,⁴⁶ and 5-O-methylvisammioside⁴⁷ reduced the levels of inflammatory factors in the serum of septic mice, including TNF-α, IL-1β, and IL-6, and inhibited the phosphorylation of NF-κB signaling pathway in the hippocampus. Gomisin N could inhibit LPS-induced mRNA levels of iNOS, COX-2, IL-1β, IL-6, and TNF-α in the paraventricular nucleus of the hypothalamus (PVN) and the central nucleus of the amygdala (CeA).⁴⁸ These findings were further validated in another study, which found that the inhibitory effect of Wuweizi (Fructus Schisandrae Chinensis) essential oil on neuroinflammation was mediated by the NF-κB/MAPK signaling pathway.⁴⁹ In addition, herbal monomers inhibited neuroinflammation in other ways. Deoxyelephantopin attenuated proinflammatory cytokine and chemokine (IL-1α, IL-1β, IL-2, IL-6, TNF-α, and CCL21) production by increasing anti-inflammatory cytokine (IL-4 and IL-10) production in the hippocampus.⁵⁰ Madecassoside can inhibit LPS-induced proinflammatory mediators in the cortex and hippocampus, such as TNF-α, IL-6, and IL-1β, by regulating the Nrf2 signaling pathway.⁵¹ The Hippo signaling pathway plays an important role in controlling inflammation, and Yes-associated protein (YAP) is the main downstream effector of this pathway. Pharmacological studies of the YAP activator XMU-MP-1 have shown that activation of YAP can induce neuroinflammation and reduce hippocampal autophagy.⁵² In addition to inhibiting NF-κB signaling pathways, resveratrol could also ameliorate LPS-induced autophagy disruption through YAP-mediated autophagy dysfunction.⁵² TCM formulas also have significant anti-neuroinflammatory effects. Jiaotaiwan significantly decreased the contents of TNF-α and IL-6 in the serum of LPS-treated experimental animals and inhibited the phosphorylation of NF-κB p65 and IκB-α.²⁵ Qiang xin 1 significantly reduced IL-1β and TNF-α levels in serum, hippocampus, and frontal cortex on days 1 and 10 after CLP surgery.²⁵ Xiaobuxin decoction (小补心汤) also inhibited LPS-induced elevated levels of IL-1β and TNF-α in the hippocampus.⁵³ In addition, a study examining whether baroreflex activation could control neuroinflammation in septic rats found that electrical stimulation of the aortic suppressor nerve attenuated LPS-induced proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in the hypothalamus, suggesting that aortic suppressor nerve stimulation could modulate neuroinflammation in heat stroke⁵⁴ (supplementary Tables 1-3).

4. MICROGLIA AND ASTROCYTE ACTIVATION

The changes and activation of microglia and astrocytes are the main manifestations of SAE.⁵⁵ The M1 microglia are classically activated and mediate the inflammatory response, secrete IL-1, IL-6, and TNF-α, and produce cytotoxic effects,⁵⁶ leading to brain damage and cognitive dysfunction.⁵⁷ Luteolin,⁵⁸ baicalin,⁵⁹ muscone,⁶⁰ and saikosaponin-d⁶¹ inhibit LPS-induced microglial activation and the overexpression of inflammatory factors, such as IL-1β, IL-6, and TNF-α, by regulating the High Mobility Group Box 1 (HMGB1)/Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. Ginsenoside Rg1 suppressed the activation of Iba1, a specific biomarker of microglia activation, and improved noncanonical beclin 1-independent autophagy in the hippocampus.⁶² Artemisinin is an antimalarial drug with strong immunomodulatory effects. Artemisinin significantly inhibited the nuclear translocation of NF-κB and proinflammatory cytokine expression by activating the adenosine monophosphate-activated protein kinase alpha 1 signaling pathway in microglia.⁶³ In addition, microglia with the M2 phenotype protect neurons by secreting neurotrophic factors. Therefore, regulating the transformation of microglia into the M2 phenotype can reduce oxidative stress injury and inflammation in the brain caused by sepsis and alleviate cognitive dysfunction.^64,65 Elevated triggering receptor expressed on myeloid cells 2 (TREM2) expression on the cell surface promotes the transformation of microglia into the anti-inflammatory phenotype (M2).⁶⁶ Yinyanghuo (Herba Epimedii Brevicornus) and Xianmao (Rhizoma Curculiginis) reversed the decreased expression of TREM2 protein in the hippocampus of LPS-treated mice; thus, EX exerted anti-neuroinflammatory effects by regulating microglial transformation and activation through the involvement of the TREM2 axis.⁶⁷ Moreover, high performance liquid chromatography-mass spectrometry analysis found that curculigoside, icariin, icariin Ⅱ, and orcinol glucoside might cross the BBB and exert neuroprotective effects after EX administration.⁶⁷ Capparis spinose extract⁶⁸ and Qiang Xin 1²⁶ also regulated the transformation of microglia from an inflammatory phenotype (M1) to an anti-inflammatory phenotype (M2).

During sepsis, activated astrocytes release a variety of inflammatory mediators, such as TNF-α, IL-1β, IL-6, and IL-18.⁶⁹ In addition, astrocyte proliferation leads to the oversecretion of eotaxin, which adversely affects learning and memory.⁷⁰ Malva sylvestris extract and Banxia Xiexin decoction (半夏泻心汤) inhibited LPS-induced increases in the number of astrocytes in the mouse brain.^71,72 Animal studies confirmed that scutellarin inhibited LPS-induced activation of microglia and astrocytes in the hippocampus. Moreover, it was further found in a cell experiment that scutellarin inhibited the release of proinflammatory factors (IL-1β, IL-6, and TNF-α) and promoted the production of an anti-inflammatory factor (IL-4) by regulating the TLR4/NF-κB pathway in astrocytes.⁷³

5. OXIDATIVE STRESS

Because of its unique characteristics, brain tissue is more vulnerable to oxidative damage during sepsis than other organs.⁷⁴ Both experimental and clinical studies have demonstrated oxidative brain damage during sepsis. An animal study found that LPS injection induced the depletion of antioxidant enzymes in the brain and plasma.⁷⁵ Another CLP-induced sepsis study found significant increases in lipid peroxidation and nitrite and superoxide (O₂⁻) levels in the brain 48 h after surgery.⁷⁶ Oxidative damage to the brain is also present in patients with sepsis. Hamed et al ⁷⁷ found that nitric oxide (NO) and lipid peroxidation levels in cerebrospinal fluid were significantly increased in patients with SAE compared with sepsis only. A variety of herbal monomers can alleviate LPS-mediated neuroinflammation and cognitive impairment by inhibiting oxidative stress. 6-Gingerol,²³ honokiol,⁷⁸ cinnamic acid,⁷⁹ scutellarin,⁸⁰ 25-methoxy hispidol,⁸¹ and imperatorin⁸² all reversed the LPS-induced increase in malondialdehyde (MDA, lipid peroxidation marker) and the decrease in superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT) in the hippocampus and prefrontal cortex.

Electroacupuncture also has antioxidative stress effects. Stimulation of the Baihui (GV20) acupoint with electroacupuncture significantly decreased hydrogen peroxide and MDA content and increased the levels of GSH and CAT in the hippocampus.²⁹ Another study investigated the protective effect and mechanism of electroacupuncture stimulation at the Baihui (GV20) and Zusanli (ST36) acupoints with different waveforms on brain injury caused by CLP. Compared with the CLP group, different waveforms of electroacupuncture could significantly reduce the content of MDA and increase the levels of SOD and CAT in the hippocampus of rats 48 h after the operation.⁸³ The comparison between different waveforms shows that the dilatational wave had the most significant effect, followed by the intermittent wave, and the continuous wave had the lowest effect.⁸³ Nuclear-related factor 2 (Nrf-2) is an important transcription factor that can induce the expression of antioxidant response genes. HO-1 is a downstream enzyme of Nrf-2 and an endogenous antioxidant enzyme. Electro-acupuncture [Quchi (LI11), Baihui (GV20), and Zusanli (ST36)] significantly increased SOD activity and decreased MDA content in the hippocampus of CLP-treated rats by upregulating Nrf-2 mRNA and HO-1 protein expression.²⁸ Moreover, the study also found that electroacupuncture could alleviate hippocampal neuron loss, increase the density of CA1 apical dendrites, and upregulate synaptophysin in CLP rats after 10 d of treatment.²⁸

6. BLOOD-BRAIN BARRIER BREAKDOWN

In a rat model of sepsis established by LPS, Evans blue (EB) dye was injected through the tail vein, and there was increased EB content in brain tissue, indicating that blood-brain barrier permeability increased during sepsis.⁸⁴ Clinically, studies have shown that magnetic resonance imaging reports of SAE patients often indicate vasogenic edema and white matter hyperintensity, which suggest BBB injury.^85,86 One possible mechanism for BBB damage is the disruption of tight junction proteins. Endotoxin and ROS destroys the structure and function of tight junction proteins.^87,88 In addition, inflammatory cytokines, for example, TNF-α, is another key factor mediating BBB injury.⁸⁹ TNF-α was found to induce actin depolymerization, resulting in the generation of intercellular gaps in endothelial cells.⁹⁰ TNF-α also activates the internalization of VE-cadherin and protein-kinase-6 to increase barrier permeability.⁹¹ By modulating the TLR4/NF-κB pathway, isoliquiritigenin,⁹² kaempferol,⁹³ and astragalus injection⁹⁴ could ameliorate the reduction in tight junction proteins in the cerebral microvascular endothelial cells of septic mice. In addition, curcumin pretreatment reduced the expression of the BBB damage indicator matrix metalloprotein 9, which confirmed that curcumin could ameliorate LPS-induced BBB damage.⁹⁵ The investigators believe that this is related to curcumin significantly increasing SOD and glutathione peroxidase activity and decreasing MDA concentration.⁹⁵

7. NEUROTRANSMITTER PATHWAY CHANGES

The normal synthesis and release of neurotransmitters are essential to ensure the conduction of "information" by neurons. During sepsis, inflammatory processes drive changes in neurotransmitter pathways, such as 5-hydroxytryptamine (5-HT).^{96,⇓,⇓-99} These changes lead to behavioral changes in patients with sepsis.^{100,⇓,⇓,⇓,⇓-105} The plasma 5-HT level was significantly increased in mice with CLP-induced sepsis, and the microvascular disorders were alleviated after administration of a 5-HT inhibitor (paroxetine), which indirectly proved that 5-HT plays a role in the pathogenesis of sepsis-induced brain dysfunction.¹⁰⁶ Ginsenoside Rb1¹⁰⁷ and paeonol¹⁰⁸ effectively reversed LPS-induced reduction in 5-HT levels in the hippocampus of mice. A metabolic study in the hippocampus of mice with sepsis found that dioscin corrected the disorder of 5-HT metabolites, including 5-hydroxyindoleacetic acid, indoleacetaldehyde, and L-tryptophan.¹⁰⁹

Neurotransmitters mediate information transmission, and neuronutrients such as brain-derived neurotrophic factor (BDNF) build and maintain nerve cell circuits. BDNF is a key factor in maintaining long-term information storage in the hippocampus.¹¹⁰ Deletion of BDNF in the hippocampus impaired spatial and aversive memories in adult mice.¹¹¹ In a mouse model of LPS-induced sepsis, BDNF levels were significantly decreased, which was accompanied by memory and behavior disorders.⁸² Therefore, upregulation of BDNF expression may have a neuroprotective effect and alleviate memory function. Ginsenoside RK1 (RK1), pseudoginsenoside HQ (PHQ) and sesquiterpenoids (SPG) are different active components derived from ginseng. RK1,¹¹² PHQ,¹¹³ SPG,¹¹⁴ liquiritigenin,¹¹⁵ geniposide,²² natural alkaloids¹¹⁶ Zhiqiao (Fructus Aurantii Submaturus),¹¹⁷ and Mahuang Fuzi Xixin decoction²⁴ could upregulate the expression level of BDNF and activate the downstream TrkB pathway, thus alleviating LPS-induced behavioral and cognitive impairment. In addition, BDNF and c-FOS are biomarkers of sleep deprivation.¹¹⁸ Senkyunolide I reversed the reduced BDNF levels and increased c-FOS levels in the cerebral cortex and hippocampus of septic mice. In contrast, sleep deprivation attenuated the ameliorative effect of senkyunolide I on CLP-induced brain dysfunction. Therefore, senkyunolide I could prevent brain dysfunction caused by inflammation and sleep loss by improving sleep.¹¹⁹

8. ABNORMAL CEREBRAL PERFUSION AND CEREBRAL MICROCIRCULATION DYSFUNCTION

Cerebrovascular autoregulation was impaired in patients with sepsis.¹²⁰ Cerebral vascular endothelial cell injury marked cortical perivascular edema, and astrocyte foot process swelling occur in CLP-induced SAE animal models.¹²¹ Similar brain microcirculation changes were also observed in LPS-induced SAE animal models.¹²² Therefore, abnormal cerebral perfusion and brain microcirculation dysfunction are closely related to the occurrence of SAE. Using the intravoxel incoherent motion (IVIM) technique, Liang et al ¹²³ found that madecassoside (MA) had beneficial effects on brain microstructure and microcirculatory perfusion in rats with LPS-induced sepsis. IVIM mainly contains two parameters: the D value (reflecting the integrity of the neural tissue) and the f value (reflecting the perfusion condition of microcirculation).^124,125 Intraventricular injection of LPS decreased the D value and increased the f value in the hippocampus and cortex of SAE rats, which suggested that there was cytotoxic edema and an increased microcirculation filling degree in the brain.¹²³ With increasing doses, MA significantly reversed the reduced D value and increased f value in the cerebral cortex and hippocampus of rats, indicating that MA alleviated cerebral microcirculation in septic rats.¹²³

9. OTHERS

In rat models, amyloid β-peptide (Aβ) accumulates significantly in the hippocampus 30 d after CLP, which is associated with cognitive impairment.¹²⁶ Similarly, Aβ accumulation in the rat hippocampus was found in a model of LPS-induced behavioral deficits during sepsis.¹²⁷ Moreover, there is clinical evidence of Aβ accumulation in the brains of patients with sepsis. Immunohistochemical analysis of brain tissue from 5 patients who died of sepsis showed diffuse staining of Aβ precursor protein.⁸⁵ Arctigenin inhibited LPS-induced increases in Aβ accumulation, amyloid precursor protein (APP) and β-site amyloid precursor protein cleavage enzyme 1 levels (BACE1), thereby significantly improving spatial learning and memory deficits in septic mice.¹²⁸ In addition, endothelial nitric oxide (eNOS) inhibitors increase Aβ levels in human microvascular endothelial cells.¹²⁹ Supplementation with NO could significantly reduce the levels of BACE1 and APP in the hippocampus of eNOS-deficient mice.¹³⁰ Electroacupuncture [Baihui (GV20) and Zusanli (ST36)] treatment significantly increased both NO and phospho-eNOS levels and reduced Aβ accumulation at 30 d after CLP. Moreover, the eNOS inhibitor (L-NAME) reversed the inhibitory effect of electroacupuncture treatment on the Aβ level, and cognitive dysfunction was not significantly alleviated in septic mice.²⁷

The NLRP3 inflammasome is a multiprotein complex that can activate pro-caspase-1, which in turn activates the secretion of IL-1β and IL-18.¹³¹ Studies have shown that the NLRP3 inflammasome is activated in neuroinflammatory states.^132,133 Fisetin treatment of SAE promoted mitophagy and ROS elimination via Pink1/Parkin signaling, blocked NLRP3 inflammasome activation, and inhibited IL-Iβ release into the CNS.¹³⁴ Similarly, scutellarin¹³⁵ and crocin¹³⁶ can also inhibit LPS-induced neuroinflammation by inhibiting the NLRP3 inflammasome.

Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor and an anti-inflammatory factor that plays an important role in regulating neuronal proliferation, differentiation, and apoptosis in the brain. LPS-induced neuroinflammation reduced FGF-2 levels.¹³⁷ However, FGF-2 suppresses the release of inflammatory cytokines.¹³⁸ Therefore, we can infer that neuroinflammation is associated with FGF-2 abnormalities. Paeoniflorin ameliorated the LPS-induced decrease in FGF-2 levels in the hippocampus, but the administration of the FGFR1 inhibitor SU5402 blocked the regulatory effects of paeoniflorin on neuroinflammation.¹³⁹ Liquiritin also increased the levels of FGF-2 in the hippocampus of septic mice.¹⁴⁰

10. CONCLUSION

Previous studies have shown that TCM formulas, some medicine plant active ingredients, and acupuncture therapy can exert peripheral anti-inflammatory and antioxidant effects on a variety of diseases. In summary, these kinds of therapy also have significant central anti-inflammatory effects. The most frequent traditional herbal remedies reported in the treatment of SAE in animal models are Renshen (Radix Ginseng), Jianghuang (Rhizoma Curcumae Longae), Huzhang (Radix Polygoni Cuspidati), Huangqin (Radix Scutellariae Baicalensis), Shengjiang (Rhizoma Zingiberis Recens), and Zhizi (Fructus Gardeniae). The reduced secretion of anti-inflammatory cytokines, activation of inflammatory response signaling pathways, and over-activation of microglia were reported to be the main mechanisms of these treatment. To date, Western medicine has limited treatment strategies for SAE, and the efficacy of antibiotics for cognitive dysfunction associated with sepsis remains to be evaluated.¹⁴¹ Complementary and alternative medicine provides more therapeutic strategies and targets for the clinical treatment of sepsis survivors. Meanwhile, more clinical studies are needed to further confirm the role of these kinds of therapy in the prevention and treatment of cognitive dysfunction associated with sepsis.

In this review, we only focused on the different mechanisms of TCM formulas, some medicine plant active ingredients, and acupuncture therapy on cognitive dysfunction associated with sepsis, and the various molecular mechanisms behind the brain injury of sepsis were not comprehensively discussed. We hope that the contents summarized in this review can help basic and clinical workers have a more comprehensive understanding of the current research status of complementary and alternative medicine in the treatment of sepsis brain injury, and bring new ideas for future basic and clinical research.

11. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://journaltcm.cn.