Neuroinflammation is a pivotal factor in the pathogenesis of many neurodegenerative diseases (1) and psychiatric illnesses like depression (2), Parkinson’s disease (PD) (3), Alzheimer’s disease (AD), (4), Huntington’s disease (5), and multiple sclerosis (6). Moreover, it has been implicated in sickness behavior (7), reduced cognition (8), and memory (9), as well as in age-related increased sensitization of the immune system to intrinsic and extrinsic stimuli (10, 11). Lastly, it has been demonstrated that the coronavirus 2019 (COVID-19) infection has triggered and reinforced obsessive and compulsive behavior leading to an aggravation of anxiety and depressive symptoms in some patients (12, 13). This could be partially explained due to an increase in the levels of proinflammatory chemokines and cytokines that are responsible for the pathophysiology of comorbid neuropsychiatric manifestations (14, 15). Also, neuropathic pain, caused by different etiology, is characterized by excessive inflammation in both the peripheral and central nervous systems (CNS), which may contribute to the initiation and maintenance of persistent pain (16). Overall, neuroinflammation can be considered as a normal response that origins in order to maintain homeostasis in the nervous system. This response involves the synthesis and release of molecules endowed with inflammatory properties; however, a neuroinflammatory process that is too strong or protracted becomes maladaptive, leading to cellular dysfunction which is seen during stress, chronic pain, and chronic neurodegeneration (17).

In this scenario, effective therapies are lacking, and a relatively dry CNS drug pipeline poses a formidable challenge to pharmacologists who have to develop drugs that can counteract the multitude of brain diseases and symptoms, such as cognitive impairment (from mild to more severe), motor impairment, memory loss, mood alterations, and pain. Indeed, the use of anti-inflammatory drugs like non-steroidal anti-inflammatory drugs (NSAIDs) can ameliorate some symptoms and reduce the extent of neurodegeneration (18); however, the primary mechanism of neuroinflammation is not well-understood and new therapeutic approaches are mandatory to provide significant relief for these patients.

Traditional medicine has been based on the use of medicinal plants for millennia, and mankind has been educated from experience and observation to use plants correctly, strengthening an extensive culture of their use. Indeed, phytotherapeutic preparations can offer a multiple approach to the multifactorial nature of neuroinflammation relying on the complex mixture of one or more plants (19).

Zingiber officinale, Centella asiatica, and Echinacea purpurea are plants rich in bioactive compounds with several pharmacological effects. Zingiber officinale Roscoe (ginger), belonging to the Zingiberaceae family, has been commonly consumed as a spice and used as herbal medicine for a long time (20). From a pharmacological point of view, ginger demonstrated to share with NSAIDs the properties of inhibiting prostaglandin synthesis and to be dual inhibitors of cyclooxygenase-2 (COX-2) and lipoxygenase (LOX) enzymes; therefore, it can effectively reduce the synthesis of both prostaglandins (PGs) and leukotrienes (LTs) (21). Additionally, it can also reduce the synthesis of proinflammatory cytokines, such as TNF-α, IL-1, and IL-8 (22, 23). These preclinical findings were supported by a study in which ginger reduced TNF-α and hs-C-reactive protein (CRP) levels in patients with diabetes (24). Moreover, ginger showed protective properties against rheumatic diseases, via rebalancing musculoskeletal disorders and joint inflammation-inducing T-helper-2 and anti-inflammatory cytokine production (e.g., IL-4 and IL-10) (25); decreasing IL-2, IL-6, and IL-1β cytokine levels; and inhibiting the release of substance P as pain and inflammation mediator (26). The beneficial effects of ginger have been mainly attributed to the presence of gingerols and shogaols (phenolic compounds) (27).

Echinacea purpurea (L.) Moench is an endemic plant in the US Great Plains and in the Canadian prairies of North America. Even though Echinacea originates in North America, the plant’s beneficial effects were discovered by Europeans who used it for its immunomodulatory properties. The bioactive constituents are mainly phenols, which are reported to exert neuroprotective effects by reducing apoptotic markers (28), polar caffeic acid derivatives (cicoric acid and echinacoside), and lipophilic alkamides (29, 30). Alkamides, in early investigations, have been demonstrated to evoke stimulatory effects on phagocytosis, and these constituents have been found in relevant concentrations in the human blood after the oral intake of Echinacea extract. Alkamides share similarities with anandamide, the endogenous ligand of cannabinoid (CB) receptors, and consequently bind to CB receptors, which are supposed to be the theoretical mechanism of action of Echinacea alkamides as immunomodulatory agents (31). In addition, Centella asiatica was successfully used for a long time in traditional Chinese medicine as a nerve tonic (32). The bioactive micronutrients are mainly asiatic acid, asiaticoside, madecassoside, and madecassic acid and are reported to be effective in retarding brain aging and assisting in the renewal of the neural tissue; therefore, it is effective in improving memory, increasing attention, and concentration (33). Centella asiatica has also shown an anti-hyperalgesic and protective profile in a rat model of osteoarthritis when the extract of this plant was intra-articularly injected (34). Despite the beneficial effects of these plants, as previously reported, no preclinical data are available about the use of a mixture of these three plants against the plethora of neuroinflammation symptoms.

The aim of this study was to highlight the efficacy of a repeated treatment with a vegetal mixture composed of Zingiber officinale, Echinacea purpurea, and Centella asiatica in a mouse model of neuroinflammation obtained by the sub-chronic injection of LPS. In particular, we focused our attention on a plethora of neuroinflammatory symptoms, such as memory impairment, depression, anhedonia, pain, and motor alterations. Moreover, ex vivo analyses were performed to determine the effect of the treatment on astrocytosis and microgliosis in the spinal cord and prefrontal cortex (PFC) and on the selected proteins of the complement system in the PFC.

Neuroinflammation was established by the sub-chronic injection of 1 mg kg^–1 LPS (for a total of four administrations performed on days 1, 3, 5, and 8). The vegetal mixture was composed of dry extracts obtained from Zingiber officinale (150 mg kg^–1), Echinacea purpurea (20 mg kg^–1), and Centella asiatica (200 mg kg^–1) and was daily per os administered from day 1 till the end of the behavioral experiments. From day 9 onward, several symptoms of neuroinflammation were analyzed. As reported in Figure 1, LPS induced a significant decrease in the animal’s pain threshold measured by thermal and mechanical non-noxious stimuli (Cold plate and von Frey tests, respectively). Indeed, on day 9, the licking latency significantly decreased from a value of about 17 s in the control group to about 9 s in the animals treated with LPS when the mice were exposed to a cold surface at 4°C (Cold plate test; Figure 1A). The daily administration of the vegetal mixture was able to counteract the thermal allodynia caused by LPS. One point to be noted is that the results were obtained 24 h after the last treatment with the mixture and were not evoked by the daily acute administration. Similarly, the paw stimulation with a non-noxious mechanical stimulus allowed us to observe a decrease in the withdrawal response in LPS-treated mice that was recovered in the group treated with LPS + vegetal mixture (Figure 1B). The LPS treatment also induced motor incoordination which was evaluated by the Rota rod test. The results showed a massive increase in the number of falls from the rod during 10 min in comparison to the vehicle + vehicle-treated animals. The combination of Zingiber officinale, Echinacea purpurea, and Centella asiatica improved the mouse performance determining a significant reduction in the number of falls, which was evident by the scores similar to those measured in the control animals (Figure 2). With regard to the impairments affecting memory and mood, we analyzed the depression-like behavior by the Porsolt test, anhedonia by the sucrose preference test, and memory by the passive avoidance test. In the Porsolt test, repeated administration of LPS caused a reduction in the mobility time after the mice were placed in a cylinder filled with water from where they could not escape. The treatment with the vegetal mixture significantly increased this parameter, reaching a value of about 150 s in comparison to 90 s observed in the LPS-treated animals (Figure 3). Moreover, the extracts were shown to reduce the amnesic effects of LPS in the passive avoidance test (Figure 4). The repeated administration of the vegetal mixture significantly antagonized the cognitive impairments induced by the lipopolysaccharide. In the retention test, the time spent in the light box was 73 ± 11 s for vehicle + vehicle group, 31 ± 4 s for LPS + vehicle group, and 103 ± 6 s for LPS + vegetal mixture-treated animals (Figure 4). Anhedonia, the incapacity to experience pleasure, is a hallmark symptom of several nervous system diseases. Anhedonia was induced by LPS injections and was evaluated as a significant reduction in sucrose intake in comparison to control animals; however, the treatment with the vegetal mixture was unable to restore this parameter (Figure 5). At the end of the behavioral measurements, the dorsal horn of the lumbar spinal cord was analyzed. Four LPS injections induced a significant increase in the number of GFAP and Iba1-positive cells (Figures 6, 7, respectively). Also, the fluorescence intensity of astrocytes and microglia was upregulated by LPS in comparison to the control group. The vegetal mixture prevented the activation of glial cells, as shown in Figures 6, 7. LPS treatment also caused a significant increase in the density of CD11b, a specific marker of microglia and macrophages, and GFAP in the PFC of the inflamed animals, which suggests that microgliosis and astrocytosis also occur in this region (Figure 8). The vegetal mixture recovered the CD11b signal to the level detected in the vehicle-treated animals (Figures 8 A,B), but did not affect the GFAP density, which was unmodified with respect to LPS-treated mice (Figures 8C,D). In recent years, the involvement of the complement system in the progression of neurodegenerative and neuroinflammatory disorders emerged in the interest of researchers. Because of the huge increase in the CD11b, which selectively binds the C3 component of the complement system, we determined whether LPS could also impact the expression of the C1q and the C3 complement proteins, i.e., those complement components that are mainly involved in the classic and the alternative pathways of the activation of the immune complex complement cascade and that are associated to the mechanisms of synaptic pruning and remodeling of central synapses (44, 45). Both C1q and C3 proteins were over-expressed in the PFC of the LPS-treated mice. The vegetal mixture significantly reduced the C3 overproduction, leaving unaltered the density of the C1q protein (Figure 9). Finally, the mixture was also able to significantly reduce the overproduction of TNF-α and IL-1β cytokines in the PFC and spinal cord, which were found to be increased in the LPS-treated animals (Figure 10).

Neuroinflammation has been implicated as a pathological contributor in several diseases affecting the nervous system, and alleviation of neuroinflammation is believed to reduce disease severity and improve patient outcomes in the majority of cases. Diseases involving the nervous system and different neurodegenerative syndromes, excluding those simply associated with aging, are currently one of the great problems in global health, and there is no doubt that neuroinflammation is involved to a lesser or greater extent in most brain pathologies, whether chronic, neurodegenerative, or acute brain damage, for a multitude of different causes. To note, recent studies have highlighted that SARS-CoV-2 infection also induces neuroinflammation with severe long-term consequences (46) due to the evoking of cytokine storm (47).

In this work, we highlighted the efficacy of a vegetal mixture composed of Echinacea Purpurea, Centella asiatica, and Zingiber officinale in a mouse model of neuroinflammation evoked by the sub-chronic injection of LPS. In particular, the mixture was able to counteract pain, depression, and motor and memory impairments, all pathologies which are associated with neuroinflammation. Moreover, ex vivo analysis demonstrated the efficacy of the treatment in reducing glial cell activation in the spinal cord and in stabilizing the central component proteins that were increased by LPS.

It has been described that systemic infection is associated with an increased frequency of behavioral and cognitive complications (48, 49). The stimulation of the peripheral immune system in mice evoked massive neuroinflammation, which results in prolonged sickness (10), impaired working memory (50), and depressive-like behavior (51).

In this context, it is crucial to design new therapies that can simultaneously act on various aspects, such as improving the recovery from symptoms, controlling the inflammatory and antioxidant processes, and modulating the immune response to attenuate the progression of the disease.

Over the years, the phytotherapeutic approach has been shown to be a valid strategy against human diseases and disorders. The plants were used for medicinal purposes since ancient times because the transcripts describe their effectiveness against various disorders. Several epidemiological studies reported a close correlation between the intake of a diet rich in antioxidant phytochemicals and a lower incidence of age-related neurological disorders for which inflammation is the core pathophysiological mechanism (52). In this study, the neuroinflammatory processes were evoked by the sub-chronic injection of LPS, a toll-like receptor 4 (TLR-4) ligand (53). TLR-4 is primarily expressed in microglia in the CNS (54), and its activation determined the production of proinflammatory cytokines, prostaglandins, and nitric oxide (NO) (55, 56), the key mediators of the neuroinflammatory processes. After four injections of LPS, mice reported mechanical and thermal allodynia as measured by the von Frey and Cold plate tests, confirming that inflammation causes an alteration in the mouse pain threshold and induces pathological hypersensitivity, which is the first step from physiological nociception to persistent pain (57). Concomitantly, animals developed behavioral symptoms predictive of mood and cognitive alterations that were largely prevented by the vegetal treatment. All the pain-relieving effects evoked by the treatment can be attributed to the complexity of the bioactive compounds present in the vegetal mixture. An important phenolic component emerged from the phytochemical characterization, in particular, Zingiber officinale extract contained gingerols (3.107%) and 6-shogaol (0.647%), which have been found to be effective in alleviating inflammation, especially in inflammatory bowel diseases (58, 59), and in reducing pain evoked by formalin and acetic acid injection in mice (60). In this context, LPS caused an overproduction of proinflammatory cytokines, such as TNF-α and IL-1β in the prefrontal cortex and spinal cord regions, which was counteracted by the vegetal mixture. A study by Ha and colleagues demonstrated the neuroprotective role of 6-shogaol in a BV-2 and primary microglial cell cultures stimulated by LPS and in an in vivo model of neuroinflammation. In particular, these effects seem to be related to both anti-inflammatory and antioxidant properties exerted by 6-shogaol on activated microglia, the resident immune cells of the nervous system (61). Centella asiatica is characterized by the presence of 2.6% phenols, mainly constituted by flavonoids and phenylpropanoid derivatives (34). This extract has already been demonstrated to possess anti-hyperalgesic properties in a rat model of osteoarthritis and reduce the NO imbalance in a cell line of macrophages stimulated by LPS, thus highlighting the antioxidant capacity of these compounds (34). In addition, Centella is enriched in polyphenols, including rosmarinic acid which possesses anti-inflammatory activities (62, 63) because of its ability to positively control the complement cascade pathways (64). Moreover, extensive literature reported the neuroprotective role of Centella asiatica. Indeed, its oral supplementation resulted in a strong memory enhancement effect in the preclinical studies (65), and the positive modulation of cognitive functions has also been confirmed in elderly volunteers (66). In an aged Alzheimer’s disease mouse model, water extract of Centella asiatica improved cognitive functions via increasing the Nrf2 gene expression in the hippocampus and by reducing Aβ plaque-associated SOD1 in the hippocampus and cortex (67).

Echinacea purpurea extract is predominantly (6%) constituted by phenolic compounds (0.037% chlorogenic acid, 3.825% cicoric acid, and 2.083% caftaric acid), which exhibit immunomodulatory and antioxidant properties (28). It is important to mention that Echinacea preparations, particularly obtained from E. purpurea, have been proposed to be used as a preventive treatment or therapeutic adjuvant against COVID-19 and as an immune-boosting agent in people at risk or with comorbidities, owing to its plausible effects on immunity, infection, and inflammation (68). Reports regarding the enhancement of immune function suggest that this effect is related to increased secretion of several cytokines by monocytes, including TNF-α, IL-1, IL-6, and IL-10 (69). Even if each extract produces effective results on its own, the mixture contains dozens of compounds with different therapeutic effects that act by different mechanisms, thus making possible a manifold approach to the complexity of the disease evoked by LPS.

The alteration in the mouse pain threshold was not the only symptom caused by LPS, but it also caused mood disorders like depression and anhedonia, and memory and motor impairments. All these alterations were counteracted by the treatment with plant extract except for anhedonia. A number of studies indicate how neuroinflammation is linked to the neurotransmitter acetylcholine which helps organisms to filter the enormous amounts of information received from the environment.

Particularly, in neuroinflammation, the α7 nicotinic acetylcholine receptor (α7 nAChR) is mostly involved, since it regulates cognitive functions and inflammatory reactions.

In the sensory cortex, it acts by fine-tuning the activity of neurons to enhance attention, which subsequently helps with learning and memory (70–72). Patients with neurodegenerative diseases exhibit a lower expression of acetylcholinesterase neurons, choline acetyltransferase, and acetylcholine synthesis, release, and re-uptake. Moreover, it is well-known how α7 nicotinic acetylcholine receptor (nAChRs) plays an important role in the management of neuropathic pain (73–75). It has been demonstrated that systematic injections of LPS reduced the density of α7 nAChRs in the brain that physiologically promote the internalization of processed Aβ forms (76, 77). Subsequently, the decrease in the density of α7 nAChR on the plasma membrane impairs Aβ metabolism and promotes the accumulation of extracellular Aβ (78, 79); this might be the mechanism by which LPS leads to neuroinflammation and cognitive impairments. It has been reported by the literature that zingiber extract restores acetylcholine levels and exhibits protective ability and cognitive enhancing properties during ethanol withdrawal (80), as well as Centella asiatica extract inhibits acetylcholinesterase, inflammation, and oxidative stress activities (81). These findings support the data obtained in this study regarding the efficacy of the vegetal mixture on all symptoms analyzed.

The important cell population in the CNS is represented by glial cells, predominantly microglia and astrocytes (82). Among their functions are the regulation of PH and ionic balance for the maintaining of homeostasis, uptake and degradation of neurotransmitters, and modulation of neuroinflammation in physiological and pathological conditions (83, 84). Several studies pointed out the pivotal role of glial cells in the generation and preservation of chronic pain (85, 86). Also, neuropathic (87) and pharmacological strategies to suppress the activity of microglial cells and astrocytes are being explored as therapies demonstrating a valid option in the management of chronic and neuropathic pain conditions (87). Although LPS may induce the activation of satellite glial cells in the dorsal root ganglia (88), the importance of glial cell activation in the spinal cord has recently been suggested (89, 90). In LPS-treated mice, we recorded an increase in the number of GFAP-positive cells and upregulation of GFAP expression, as well as an increase in the number of Iba1-positive cells and upregulation of Iba1 expression. The maladaptive plasticity of the glial cells in the spinal cord was attenuated by the treatment with the vegetal mixture, thus highlighting the importance of the inhibition of these cell populations in the control of neuroinflammation. Astrocytosis and microgliosis also emerged in the PFC and could be predictive of amnesic symptoms observed in LPS-treated mice. Activated microglia and astrocytes are also central sources of the complement proteins, particularly C1q and C3 proteins, which in physiological conditions regulate neuronal development by suppressing weak synapses, but if aberrantly expressed, exacerbate synaptic derangements and central inflammation, contributing to the cognitive and mood decline associated with aging or to neurodegenerative disorders, i.e., Alzheimer’s disease (91, 92).

In this context, a significant reduction of the C3 density in the PFC of the LPS-treated mice receiving the mixture could be predictive of anti-inflammatory activity and the amelioration of the cognitive performances observed in in vivo studies. The positive impact of the vegetal mixture is of even more interest considering the concomitant persistence of a high level of C1q, which if over-expressed in the absence of other complement components modulates the gene expression critical for neuronal survival (93).

In conclusion, the present work demonstrated the efficacy of daily treatment with a vegetal mixture composed of Zingiber officinale, Centella asiatica, and Echinacea purpurea extracts in the control of neuroinflammation induced by LPS in mice. In particular, mood disorder, pain, and motor and cognition impairments were counteracted. Moreover, the vegetal mixture induced a reduction in the activation of glial cells in the spinal cord and PFC. The results obtained in this study represent an important milestone for the use of phytotherapies in the control of neuroinflammation.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Formal approval to conduct the experiments described was obtained from the Animal Subjects Review Board of the University of Florence.

LM, CG, and LD: conceptualization. CP, JL, EL, and AT: methodology. CC and VF: software. LM and EL: validation. GO: formal analysis. MB and DC: resources. LM and AP: data curation. LuM: original draft preparation. LuM, CG, LD, SP, and AP: reviewing and editing. CG, AP, and LD: supervision. CG and LD: project administration. CG: funding acquisition. All authors have read and agreed to the published version of the manuscript and have contributed substantially to the work reported.