The Effects of Andrographis paniculata (Burm.F.) Wall. Ex Nees and Andrographolide on Neuroinflammation in the Treatment of Neurodegenerative Diseases

Neurodegenerative diseases (NDs) affect millions of people worldwide, and to date, Alzheimer’s and Parkinson’s diseases are the most common NDs. Of the many risk factors for neurodegeneration, the aging process has the most significant impact, to the extent that it is tempting to consider neurodegenerative disease as a manifestation of accelerated aging. However, genetic and environmental factors determine the course of neurodegenerative disease progression. It has been proposed that environmental stimuli influence neuroplasticity. Some clinical studies have shown that healthy lifestyles and the administration of nutraceuticals containing bioactive molecules possessing antioxidant and anti-inflammatory properties have a preventive impact or mitigate symptoms in previously diagnosed patients. Despite ongoing research efforts, the therapies currently used for the treatment of NDs provide only marginal therapeutic benefits; therefore, the focus is now directly on the search for natural products that could be valuable tools in combating these diseases, including the natural compound Andrographis paniculata (Ap) and its main constituent, andrographolide (Andro). Preclinical studies have shown that the aqueous extract of Ap can modulate neuroinflammatory and neurodegenerative responses, reducing inflammatory markers and oxidative stress in various NDs. Therefore, in this review, we will focus on the molecular mechanisms by which Ap and Andro can modulate the processes of neurodegeneration and neuroinflammation, which are significant causes of neuronal death and cognitive decline.


Introduction
Neurodegenerative diseases (ND) exhibit specific epidemiological and symptomatologic characteristics and are diagnosed using separate laboratory and neuroimaging tests; the neuropathological mechanisms and treatments are also different. However, all neurodegenerative diseases can cause morbidity and reduced cognitive capacity in elderly individuals worldwide [1][2][3][4]. Alzheimer's disease (AD) accounts for nearly 80% of all

Molecular Mechanisms Involved in the Onset of Neurodegenerative Diseases
Neurodegenerative diseases differ in the accumulation of specific proteins and in the different anatomical areas in which the pathological neuronal variation is observed, but they share many of the pathological mechanisms, such as proteotoxic stress and abnormalities in the resulting catabolic systems, that lead to neuronal dysfunction and death. Indeed, in neurodegenerative diseases, the ubiquitin-proteasome system and the autophagy/lysosomal system are found to be altered. In addition, oxidative stress, apoptotic processes, and neuroinflammation play a crucial role in all neurodegenerative diseases [25]. The first evidence of an ongoing inflammatory phenomenon in AD was described many years ago [26], and several subsequent studies have also documented an active inflammatory process in PD, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and a growing number of other nervous system diseases. In these disorders, inflammation is not the trigger for the disease process, but rather, as revealed by studies using animal models, an inflammatory response involving the microglia and astrocytes, which may contribute to the process of disease progression. Therefore, the hypothesis that inhibition of the inflammatory response may be a viable strategy to reverse or slow the course of the disease is well-founded. In the brain, the immune response is performed mainly by the microglia, which influence neurons, astrocytes, and glial cells that perform a supporting function. Microglia typically only become activated to produce inflammatory and neurotrophic factors in order to repair tissue damage induced by an injury or microorganism once the immune system has been activated under pathological conditions. However, a sustained inflammatory state shows the persistence of the inflammatory stimulus and the failure to resolve the damage. Thus, the inflammatory response that physiologically represents the initiation of a beneficial process, i.e., for tissues to ensure the removal of cellular debris, can, under certain conditions, become a risk factor. Prolonged and uncontrolled inflammation can lead to the production of neurotoxic factors capable of inducing significant tissue pathology.
The expression of genes involved in amplifying inflammatory responses results when cells perceive evidence of infection or injury. Inflammation that occurs in response to infectious agents usually begins because of the activation of recognition receptors such as toll-like receptors (TLRs), which recognize certain pathogen-associated molecules that are not present in the host. The cells most involved in the innate immune response, such as macrophages and microglia, highly express these receptors. In recent years, the hypothesis that these recognition receptors may also respond to endogenous molecules, such as Nutrients 2023, 15, 3428 3 of 17 those released during necrotic processes, has been confirmed [27,28]. Purinergic receptors are also expressed on microglia and astrocytes and come into play in some pathological states [29,30].
As mentioned above, the pathological brain features of AD include extracellular amyloid plaques; in addition, the inflammatory response in AD induces an increase in the number, size, and motor activity of microglia, as well as a morphological alteration, whereby they change from branched (resting) to amoeboid (active). In addition, the microglia surrounding the plaques appear to be positive for activation markers and proinflammatory mediators, including MHC class II, COX-2, TNF-α, and some interleukins [31][32][33][34]. Proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, can act directly on the neurons to induce apoptosis [35,36], and the activation of caspases and signal-dependent transcription factors, such as NF-κB and AP-1, leads to the production of many amplifiers (e.g., IL-1β, TNF-α, IL-6). Finally, communication between the neurons and the glia can amplify the production of neurotoxic factors that contribute to AD pathology; molecules with a proinflammatory effect on the neurons can increase Aβ production and microglia-mediated inflammation [37].
The inflammatory reaction, characterized by a discharge of chemokine and ROS, is centrally triggered in several acute brain diseases, including ischemic stroke, leading to neuroinflammation, which is often followed by necrosis and apoptosis.
Immune mediators play a key proinflammatory role, enhancing brain cells and promoting the penetration of inflammatory cells such as macrophages, T cells, and neutrophils into the area affected by the ischemic insult. Cytokines such as IL-6 and TNF-α appear to be crucial mediators of post-ischemic inflammation [38].

Limitations of Current Therapies Used for Treating Neuroinflammation and Neurodegenerative Disorders in the Early Stages of the Disease
Numerous risk factors and diseases can be considered as predisposing factors for neurodegenerative diseases, including cerebrovascular disease, hyperlipidemia, smoking, diabetes, obesity, and traumatic brain injury. In contrast, protective factors are less numerous and include increased cognitive reserve, consumption of a Mediterranean diet, and regular exercise [6,39,40]; however, to date, there are no specific completely effective therapies [41]. Currently, pharmacological treatments have been proven to change the course of AD, although therapies targeting aspects of both amyloid and tau protein are under active investigation. Thus, the clinical management of AD aims to improve patient symptoms and optimize quality of life. Acetylcholine (ACh), a widely distributed neurotransmitter in the body known to improve cognition, is reduced in patients with AD; in fact, the increase in the ACh level induced by the use of acetylcholinesterase inhibitors (e.g., donepezil, rivastigmine, and galantamine) has been associated with improved cognitive ability [42][43][44][45]. Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist capable of acting by suppressing glutamate-mediated excitotoxicity, has been shown to reduce clinical deterioration in patients with moderate-to-severe AD compared with the controls [46][47][48], but this is not the case in patients with mild disease [49]. Combining acetylcholinesterase inhibitors and memantine may have marginal benefits over single-drug treatment [50]. In addition, it is necessary to consider the modest benefits of these treatments and the potential side effects of each treatment option. Even in the current treatment of Parkinson's disease, available drug therapies do not represent a complete resolution of symptoms; while they often offer reasonable control of motor symptoms [51,52], they do not alter the evolution of the disease [53]. Drug therapies targeting the motor features of Parkinson's disease act by potentiating dopamine signaling, involving the direct implementation of the neurotransmitter dopamine (e.g., levodopa), monoamine oxidase (MAO) enzyme inhibition (e.g., selegiline), and catechol-O-methyltransferase (COMT) inhibition (e.g., entacapone). Anticholinergics (e.g., trihexyphenidyl and benztropine) are effective in patients with a predominant tremor phenotype, and their use is preferred in the early stages of Parkinson's disease. Deep brain stimulation (DBS) can relieve motor fluctuations and tardive dyskine- sias in patients with drug-refractory PD, providing benefits for tremor and rigidity, but gait, balance, and cognitive abilities are unlikely to improve as a result of this treatment [54]. Electrodes placed in the inner globe pallidum or subthalamic nucleus regulate abnormal neural impulses, thereby alleviating motor symptoms [55][56][57][58]. Co-treatment with DBS may reduce the dose or adverse effects of medications, but the possibility of some complications, such as bleeding and infection, should be considered here [59][60][61]. The psychiatric manifestations of AD sometimes respond to symptomatic treatments for AD but often require more specific treatment with psychiatric medications. Depression, for example, is often treated with selective serotonin reuptake inhibitors (SSRIs) with low anticholinergic properties (e.g., citalopram, escitalopram, and fluoxetine) [62]. Treatment with neuroleptic drugs should be avoided because they are largely ineffective, and at the same time, risky because of the resulting side effects and increased risk of mortality; however, when nonpharmacological treatments yield insufficient results, the use of these drugs becomes necessary [63]. In DLB and PD, dopaminergic therapy is used to counteract extrapyramidal symptoms, even though this method provides reduced symptomatic improvement than that observed in PD. Depression and anxiety in DLB and PD can be treated with SSRIs or serotonin-norepinephrine reuptake inhibitors (SNRIs). Atypical antipsychotics may be useful for psychiatric symptoms, but should be used with great caution due to their adverse effects on movement and cognition. Traditional neuroleptics should be avoided because of neuroleptic hypersensitivity in patients with DLB. Disease-modifying agents are not yet clinically available [64]. Furthermore, treatment with dopamine agonists can cause side effects in the Parkinsonian patient, which also include changes in behavior, probably because of overstimulation of the mesolimbic dopamine receptors, in predisposed subjects, and this may force the interruption of dopamine (DA) therapy or its substitution with alternate drugs [65].

The Potential Role of Andrographis paniculata in Treating Neuroinflammation and Neurodegenerative Diseases
Despite research efforts, treatments currently used for neurodegenerative diseases, as previously noted, only provide marginal symptomatic benefits. In recent years, research has focused on products of natural origin, such as phytocompounds, that could be valuable tools in combating various diseases [66][67][68][69][70]. Among the most studied medicinal plants, Andrographis paniculata (Ap) (Burm.F.) Wall. ex Nees is known for its active phytochemical content. It is mainly constituted of high amount of alkaloids (63.75%), and flavonoids (23.01%), a moderate amount of tannins (less than 5%), and traces of phenols, saponins, glycosides, steroids, terpenoids, anthocyanins, and carotenoids. These phytochemicals can act individually or in synergy to produce an antiviral, antimicrobial, antitumor, anti-inflammatory, and analgesic effect [71,72]. The analysis of phytochemicals contained in Ap extracts, performed by Koteswara et al., reported that in addition to the previously known compounds (e.g., andrographolide, β-sitosterol, cinnamic acid, caffeic acid, ferulic acid, chlorogenic acid, etc.; and 14-deoxy-11, 12-didehydroandrographolide, neoandrographolide, andrographoside, and 14-deoxyandrographolide 7-methyldihydroogonin, etc.), they also discovered two new flavonoids: (2S)-5,7,20,30-tetramethoxyflavanone and 5-hydroxy-7,20,30-trimethoxyflavanone, characterizing them using 2D NMR spectroscopy to highlight their antioxidant activity. Andrographolide (Andro) is one of the main constituents extracted from Ap leaves. Andro, a diterpene lactone, has attracted particular interest [73][74][75][76][77]. Ap is a plant of Asian origin, known since ancient times by the name "King of Bitters". Ap, an annual shrub in the family Acanthaceae, is used in traditional Chinese medicine as an antidote for poisonous insect stings and snake bites and to treat dyspepsia, flu, and dysentery. In preclinical studies, the aqueous extract of Ap was able to reduce the expression of inflammatory markers such as TNF-α, IL-1β, IL-6 and oxidative stress makers such as ROS and thiobarbituric acid reactive substances (TBARS) with an enhancement of SOD (superoxide dismutase), CAT (catalase), and GSH (glutathione) activity [78]. In addition, pharmacokinetic analyses conducted on Wistar rats treated with Ap and Andro extract showed that the compounds could easily cross the blood-brain barrier and generate effects at the neuronal level because of their capacity for high distribution in the brain [79]. Especially in the hippocampus, Ap can reduce the activity of LPS-induced cholinesterases, thus exhibiting anti-inflammatory activity and improving mnemonic processes, as confirmed by spatial learning tests [78]. The role of Andro and its derivatives has also been studied in the context of neuroinflammation. Kumar et al., in 2020, and Zhang et al., in 2021, published two review articles concerning Andro derivatives, focusing on alterations in the different functional groups in regards to changes in biological activity. By carefully reviewing the literature from recent years and individually examining the studies performed on each derivative, they reported a collection of the chemical structures of all the derivatives, defining for each the specific biological activity observed in the various in vivo and in vitro study models [80,81]. Figure 1 summarizes the derivatives that can improve inflammation and neuroinflammation by reporting their structure formulas. expression of inflammatory markers such as TNF-α, IL-1β, IL-6 and oxidative stress makers such as ROS and thiobarbituric acid reactive substances (TBARS) with an enhancement of SOD (superoxide dismutase), CAT (catalase), and GSH (glutathione) activity [78]. In addition, pharmacokinetic analyses conducted on Wistar rats treated with Ap and Andro extract showed that the compounds could easily cross the blood-brain barrier and generate effects at the neuronal level because of their capacity for high distribution in the brain [79]. Especially in the hippocampus, Ap can reduce the activity of LPS-induced cholinesterases, thus exhibiting anti-inflammatory activity and improving mnemonic processes, as confirmed by spatial learning tests [78]. The role of Andro and its derivatives has also been studied in the context of neuroinflammation. Kumar et al., in 2020, and Zhang et al., in 2021, published two review articles concerning Andro derivatives, focusing on alterations in the different functional groups in regards to changes in biological activity. By carefully reviewing the literature from recent years and individually examining the studies performed on each derivative, they reported a collection of the chemical structures of all the derivatives, defining for each the specific biological activity observed in the various in vivo and in vitro study models [80,81]. Figure  1 summarizes the derivatives that can improve inflammation and neuroinflammation by reporting their structure formulas. A study performed in vivo by LPS-induced neuroinflammation showed that treatment with Andro reduced cortical levels of certain chemokines, such as CCL2 and CCL5. The same authors observed the anti-inflammatory effect of Andro in vitro by inducing the neuroinflammation of astrocytes using both LPS and IL-1β. In the LPSinduced neuroinflammation model, Andro treatment was able to inhibit the LPS-induced TNF-α release through the inhibition of NFkB and JNK, additionally increasing the nuclear factor erythroid 2-related factor 2 (NRF2) levels and heme oxygenase-1 (HO-1) expression through the p38MAPK and ERK-dependent signaling pathway [82]. Additionally, in the IL-1β-induced neuroinflammation model, the compound was able to reduce the release of CCL5, phosphorylation of NFkB p65 and IkBα, and glial fibrillary acidic protein (GFAP) [83]. To investigate the role of Andro in LPS-induced neuroinflammation and memory, Das et al. evaluated the expression of TLR4 and its A study performed in vivo by LPS-induced neuroinflammation showed that treatment with Andro reduced cortical levels of certain chemokines, such as CCL2 and CCL5. The same authors observed the anti-inflammatory effect of Andro in vitro by inducing the neuroinflammation of astrocytes using both LPS and IL-1β. In the LPS-induced neuroinflammation model, Andro treatment was able to inhibit the LPS-induced TNF-α release through the inhibition of NFkB and JNK, additionally increasing the nuclear factor erythroid 2-related factor 2 (NRF2) levels and heme oxygenase-1 (HO-1) expression through the p38MAPK and ERK-dependent signaling pathway [82]. Additionally, in the IL-1β-induced neuroinflammation model, the compound was able to reduce the release of CCL5, phosphorylation of NFkB p65 and IkBα, and glial fibrillary acidic protein (GFAP) [83]. To investigate the role of Andro in LPS-induced neuroinflammation and memory, Das et al. evaluated the expression of TLR4 and its endogenous ligand high mobility group box 1 (HMGB1) in primary mixed glial cells (PMLCs) in adult prefrontal cortices. Andro treatment was successful in reducing TLR4 expression and LPS-induced p-NFκB-p65 activation. In the microglia, however, a reduction in CD-68 expression levels and an increase in arginase-1 levels were observed. However, at the cortical level, Andro was able to reduce the expression of GFAP, exerting a neuroprotective action in the astrocytes through the inhibition of the release of pro-inflammatory molecules such as iNOS, COX-2, nitrates, NLRP3, and caspase-1. Finally, in cognitive memory processes, Andro antagonized the overexpression of PKC and the phosphorylation of cAMP-responsive element binding protein (CREB), which usually results in upregulated cognitive deficits. The same authors also observed that at the level of the prefrontal cortex, Andro reduced the levels of β-amyloid, APP, ptau, BACE-1 (β-secretase-1), as well as the activation of the proapoptotic caspase-3 and bax genes, and increased the levels of the anti-apoptotic gene Bcl-2, improving neuronal viability. At the hippocampal level, an increase in postsynaptic density protein 95 (PSD-95) and synapsin was observed, resulting in increased neuronal plasticity, reducing the expression levels of inflammatory cytokines such as TNF-α and macrophage inflammatory protein-1 (MIP-1), and an increasing those of TGF-β and IL-10 [84]. In another study conducted by Adedayo et al., the effect of Andro in counteracting the amnesic effect of scopolamine, like that found in AD, was evaluated. Rats treated with the aqueous extract of Ap showed an improvement in cognitive function, as assessed by the Y-Labyrinth Test and the Morris Water Maze (MWM) Test, and additional analyses showed significant reductions in AChE and Buttiril-ChE, as well as a reduction in some MAOs and purinergic enzymes such as ATPdase and ADPdase, while the levels of 5-nucleotidase and adenosine deaminase were found to be increased at the hippocampal level [85].
Because neuroinflammation also plays a crucial role in diseases such as depression and schizophrenia [86,87], some studies aimed to investigate the possible role of Andro in these diseases. In an in vivo model of schizophrenia induced by the administration of phencyclidine (PCP), Andro reduced the levels of IL-1β and TNF-α, p-p65, p-IκBα, p-p38, and p-ERK1/2 in the prefrontal cortex; improved oxidative stress through the activation of antioxidant enzymes such as SOD, CAT, and GSH-Px; and increased the levels of NRF-2, HO-1, and NAD(P)H Quinone Dehydrogenase 1 (NQO-1). Cognitive improvement was also confirmed by behavioral tests that showed increased locomotor activity [88]. Andro was also found to be effective in the treatment of stress-induced depression. Mice subjected to chronic unpredictable mild stress (CUMS) and treated with Andro showed reduced levels of NO, iNOS, IL-1β, IL-6, TNF-α, COX-2, p-p65, p-IκBα, and the NLR family pyrin domain containing 3 (NLRP3) in the prefrontal cortex, compared with the levels in untreated mice. In addition, Andro exerted a pro-autophagic action, through increased Beclin-1 expression, and decreased p-mTOR [89].
A recent study has evaluated Andro's effects in the treatment of neurological disorders induced by heavy metal exposure. Specifically, the effects of Andro on aluminum intoxication in Drosophila were assessed. Interestingly, Andro treatment on flies improved survival, locomotor performance, and learning and memory through reduced AChE and MAO activity and increased catalase activity [90].
In 2005, Iruretagoyena et al. also observed the protective action of Andro in the treatment of Multiple Sclerosis, an autoimmune neurodegenerative disease in which immune defenses, particularly lymphocytes, attack nervous system components. Indeed, in dendritic cells (DCs) pulsed with hen's egg ovalbumin (OVA), Andro counteracted the generation of peptide-MHC complexes required for T-cell activation by inhibiting the upregulation of maturation markers I-Ab, CD40, and CD86 in LPS-treated dendritic cells. Andro treatment was also tested in vivo on C57BL/6 mice with experimental autoimmune encephalomyelitis (induced with the peptide MOG35-55), and it significantly reduced the incidence of the disease, as also shown by the reduced production of IFN and IL-2 [91]. Therefore, based on the data reported in the literature, we can state that both Ap and Andro are effective in treating the inflammatory and degenerative effects that occur in the nervous system during various disease states [75,78,92,93] (Table 1). Here, we will examine these effects on diseases involving the nervous system, focusing on those most prevalent in the world population. LPS-induced reduction of TLR4 expression and p-NFκB-p65 activation; reduction in the levels of inflammatory cytokines, such as TNF-α and MIP-1 (macrophage inflammatory protein-1); increase in anti-inflammatory cytokines, such as TGF-β and IL-10; reduction of pro-inflammatory molecule expression, such as iNOS, COX-2, nitrates, NLRP3, caspase-1

Effects in aluminum poisoning
Improved survival, locomotor performance, learning, and memory through a reduction of AChE and MAO activity and increased catalase activity

Effects on Multiple Sclerosis
Prevention of the generation of peptide-MHC complexes required for T cell activation; inhibition of upregulation of maturation markers I-Ab, CD40, and CD86 in LPS-treated dendritic cells

Alzheimer Disease
In vitro studies showed that Andro was able to activate α-secretase, which is involved in the inhibition of Aβ formation, and to inhibit β-secretase, which is involved in Aβ formation, without the protective treatment causing toxicity [94]. On hippocampal neuronal cells of HT-22 mice, treatment with Andro increased the levels of nuclear factor erythroid-derived 2-like 2/Kelch-like ECH-associated protein 1 (NRF2/Keap), the antioxidant response element (ARE) gene, and the HO-1 enzyme. Since these factors are major players in anti-inflammatory and antioxidant responses, it is evident that Andro induced a cytoprotective response in the brain [95]. On mouse microglia BV-2 (microglial cells derived from C57BL/6 murine), Andro reduced the expression of Aβ, improving neuronal viability, and through the NFkB-mediated signaling pathway, reduced the levels of TNF-α, COX-2, and PGE2, i-NOS, NO, and cytokines such as IL-1β, IL-6, protecting the neurons from damage produced by inflammation [96].
In addition to Andro, its analogs have also been used on these same cells, achieving complementary results. Indeed, the analogs have shown the ability to inhibit LPS-induced NO production and iNOS expression, as well as TNF-α and IL-6 production [97].
One enzyme known to be involved in learning and memory processes, and especially in tau protein phosphorylation and increased β-amyloid production, is glycogen synthase kinase-3β (GSK3β) [98]. Andro treatment of primary hippocampal neuronal cultures induced the inhibition of the GSK3β enzyme and the reduction of its active form [99]. The same treatment conducted on human embryonic kidney (HEK293) cells showed comparable results. Because GSK3β is involved in the Wnt/β-catenin signaling pathway, known for its role in neurogenesis, the effect of Andro on this pathway was also investigated [100]. Andro treatment on hippocampal cells showed an induction of Wnt gene transcription by restoring its proper activity [99]. Several studies regarding the effects of Ap have also been conducted in mouse models. Various transgenic mouse models have been used in the literature to reproduce the features of Alzheimer's disease [101,102].
In one study in which Andro was tested on 2-month-old mice exhibiting mutations on the genes encoding for APP and presenilin (PS1), known proteins involved in AD, increased expression of the Wnt/β-catenin signaling pathway was observed [103]. In another experiment, in which 7-12-month-old mice were used, Andro was able to alter the maturation of amyloid plaques in the cortex and the hippocampus; in the early stages of the disease, the number of plaques was found to be reduced. Andro was also observed to exert its effects on tau protein phosphorylation, leading to a significant increase in post-synaptic proteins, including Shank, GluN2B, GluA2, PSD-95, as well as an increase in the inactive form of GSK3β. Moreover, behavioral tests found that Andro-treated mice showed improved learning and lower latency values, as well as improved spatial memory performance [104]. In addition to the transgenic mouse models, some work has also been conducted on Octodon degus mice, which can naturally reproduce the neurodegeneration and neurological signs of Alzheimer's with advancing age [102]. Rivera et al. performed several behavioral tests on these 12-and 56-month-old rodents to assess their degree of spatial learning and memory after Andro administration. The results obtained showed neuroprotective effects that resulted in the recovery of memory and learning, enhancement of the excitatory postsynaptic field potential (fEPSP), protection of specific proteins, such as synaptophysin (SYP), along with increased vesicular glutamate transporter 1 (vGluT1) and NMDA receptor subunit GluN2A. Their results also showed decreased phosphorylated tau protein and Aβ aggregate maturation in aged mice [105]. In contrast, on these same animals, Lindsay et al. reported the neuroprotective effects of Andro through a reduction in Aβ, GFAP, IL-6, COX-2, and oxidative stress markers such as 4-Hydroxynonenal (4-HNE) () and N-Tyrosin (N-Tyr) in the brain [106].
It is well known that impaired glucose metabolism at the neuronal level may be related to several neurodegenerative diseases, including AD [107][108][109][110][111][112]. Cognitive deficits and symptoms comparable to AD are also reproducible following the intracerebroventricular administration of streptozotocin (SZT).
Rats that underwent this treatment and were treated with Andro show improved spatial memory according to the results of the Morris Water Maze (Morris Water) and the Elevated Plus Maze tests, compared with the results for the untreated group of animals. Andro also reduced levels of neuroinflammatory markers such as TNF-α, IL-1B, and IL-16, decreasing levels of the neurotransmitter glutamate (GLU), and increasing levels of GABA. Because STZ administration induces increased levels of AChE and ptau, the group of animals that had received Andro treatment showed a significant reduction in the expression levels of AChE and ptau, as well as a reduction in oxidative stress through the attenuation of Nutrients 2023, 15, 3428 9 of 17 MDA (malondialdehyde) and nitrite, along with increased levels of GSH, SOD, and catalase compared with the levels in the group treated with STZ alone [113]. Comparable results were also obtained for diabetic rats given oral administrations of STZ and subsequently treated with Andro [114]. In another study on rat primary hippocampal neurons, treatment with Andro resulted in increased glucose uptake through increased GLUT translocation and increased ATP production, promoting AMPK-dependent glycolysis [115] (Figure 2).

Parkinson's Disease
As already widely described, inflammation and apoptosis are essential factors in various neurodegenerative diseases, of which Parkinson's is one [116,117]. To study the effects of Andro on this disease, rat midbrain glia cultures were pre-and post-treated concomitantly with LPS-induced dopaminergic neurodegeneration. The results obtained showed that Andro was able to attenuate LPS-induced dopaminergic neurodegeneration by reducing the activation of microglia and inflammatory factors such as ROS, TNF-α, NO, and PGE2. In addition, pretreatment with Andro on BV2 microglia cells reduced the expression of COX-2 and iNOS. However, in this work, the neuroprotection of Andro on neurodegeneration induced by 1-methyl-4-phenyl-pyridine (MPP), a metabolite of MPTP used to generate Parkinson's, was also tested, although pretreatment with Andro failed to reduce neuronal damage [118]. In another study conducted on mice subjected to intraperitoneal administration of MPTP, treatment with Andro produced positive results for all behavioral tests performed, including the catalepsy, grip strength, and, rotarod tests, leading to improved motor conditions [89]. In vitro, it was seen that LPS-and MPPinduced microglia activation was reduced following Andro administration, resulting in decreased NLRP3 inflammasome activation, and these data were confirmed by inhibition of the microglial expression marker Iba-1, both in vitro and in vivo, in the mouse brain [119,120]. Andro has also been shown to improve mitochondrial dysfunction through the inhibition of ROS formation and maintenance of mitochondrial membrane potential (in vitro) by additionally promoting autophagosome formation and the elimination of damaged mitochondria [120]. The neuroprotective effects of Andro on MPP-induced damage were also studied on neuroblastoma cells (SH-SY5Y). In these cells, andrographolide-lipoic acid (AL-1) conjugate protected against MPP-induced damage, leading to increased cell viability and the inhibited phosphorylation of NF-κB p65 and IκBα. Moreover, in MPTP-treated mice, AL-1 protected against the loss of TH-positive dopaminergic neurons in the substantia nigra pars compacta, attenuated dopamine loss in the striatum, and improved motor functions, as showed by behavioral tests [121].
However, in a later study performed in SH-SY5H cells, Ketterman et al. observed that the administration of Andro did not provide protective effects against oxidative stress [119].

Parkinson's Disease
As already widely described, inflammation and apoptosis are essential factors in various neurodegenerative diseases, of which Parkinson's is one [116,117]. To study the effects of Andro on this disease, rat midbrain glia cultures were pre-and post-treated concomitantly with LPS-induced dopaminergic neurodegeneration. The results obtained showed that Andro was able to attenuate LPS-induced dopaminergic neurodegeneration by reducing the activation of microglia and inflammatory factors such as ROS, TNF-α, NO, and PGE2. In addition, pretreatment with Andro on BV2 microglia cells reduced the expression of COX-2 and iNOS. However, in this work, the neuroprotection of Andro on neurodegeneration induced by 1-methyl-4-phenyl-pyridine (MPP), a metabolite of MPTP used to generate Parkinson's, was also tested, although pretreatment with Andro failed to reduce neuronal damage [118]. In another study conducted on mice subjected to intraperitoneal administration of MPTP, treatment with Andro produced positive results for all behavioral tests performed, including the catalepsy, grip strength, and, rotarod tests, leading to improved motor conditions [89]. In vitro, it was seen that LPS-and MPP-induced microglia activation was reduced following Andro administration, resulting in decreased NLRP3 inflammasome activation, and these data were confirmed by inhibition of the microglial expression marker Iba-1, both in vitro and in vivo, in the mouse brain [119,120]. Andro has also been shown to improve mitochondrial dysfunction through the inhibition of ROS formation and maintenance of mitochondrial membrane potential (in vitro) by additionally promoting autophagosome formation and the elimination of damaged mitochondria [120]. The neuroprotective effects of Andro on MPP-induced damage were also studied on neuroblastoma cells (SH-SY5Y). In these cells, andrographolide-lipoic acid (AL-1) conjugate protected against MPP-induced damage, leading to increased cell viability and the inhibited phosphorylation of NF-κB p65 and IκBα. Moreover, in MPTP-treated mice, AL-1 protected against the loss of TH-positive dopaminergic neurons in the substan-tia nigra pars compacta, attenuated dopamine loss in the striatum, and improved motor functions, as showed by behavioral tests [121].
However, in a later study performed in SH-SY5H cells, Ketterman et al. observed that the administration of Andro did not provide protective effects against oxidative stress [119].
Dysregulation of GLU levels is involved in several processes that may cause neuronal damage [122][123][124]. In this regard, in 2014, Yang et al. studied the neuroprotective effect of Ap extract on HT22 neuronal cells in the mouse hippocampus following glutamate-induced damage. The results showed that Ap can significantly reduce GLU-induced neuronal mortality and cytosolic lactate dehydrogenase (LDH) levels, Ca 2+ influx, and intracellular ROS production induced by GLU in a dose-dependent manner. In addition, the same authors also observed a significant decrease in the phosphorylation of MAPK, p38, ERK, and JNK. At the same time, Western blotting analysis revealed a restoration of the levels of anti-apoptotic proteins such as Bcl-2, Bid, and Bax, and a reduction in apoptosis-inducing factor (AIF) after Andro treatment [95] (Figure 3). induced damage. The results showed that Ap can significantly reduce GLU-induced neuronal mortality and cytosolic lactate dehydrogenase (LDH) levels, Ca 2+ influx, and intracellular ROS production induced by GLU in a dose-dependent manner. In addition, the same authors also observed a significant decrease in the phosphorylation of MAPK, p38, ERK, and JNK. At the same time, Western blotting analysis revealed a restoration of the levels of anti-apoptotic proteins such as Bcl-2, Bid, and Bax, and a reduction in apoptosis-inducing factor (AIF) after Andro treatment [95] (Figure 3).

Brain Ischemia-Reperfusion Injury
Cerebral ischemia is a pathological condition characterized by a reduced blood supply that can affect different brain areas and induce significant neuronal damage [125]. In a study conducted on mouse brain endothelial cells, no positive effects were found following the administration of Andro, as the compound not only failed to improve the viability of these cells, but also induced a marked reduction in the number of neuronal cells due to increased LDH release and increased apoptosis, blocking cell growth in the G0/G1 phase [126]. However, from many other studies, especially those conduced in recent years, the protective action of Andro on ischemic damage seems to emerge and to be confirmed. More specifically, in a rat-mouse model with permanent middle cerebral artery occlusion (pMCAO), Andro reduced the infarct volume and microglia activation in peri-infarct areas by combatting increased levels of inflammatory markers such as IL-1β, TNF-α, and PGE2 and the transcription factor NFkB [127]. In another study, however, rats undergoing ischemic brain injury/reperfusion (CI/R) treated with Andro showed decreased cerebral infarction, reduced superoxide anion and nitrotyrosine, reduced expression of gp91phox/NOX2, IL-1β and hypoxia inducible factor (HIF), as well as reduced NFkB p65, thus confirming the protective effects of Andro in counteracting an ischemic attack [73]. Mice with CI/R-induced oxidative brain damage and treated with Andro also reported a reduced magnitude of infarction and subsequent neurological deficits, accompanied by reduced free radical production, as well as reduced nitrotyrosine, CD11b, NOX2, and iNOS formation [128].
Another andrographolide derivative, triacetylandrographolide (CX-10), has been used to test for possible neuroprotective effects against cerebral ischemia. On the

Brain Ischemia-Reperfusion Injury
Cerebral ischemia is a pathological condition characterized by a reduced blood supply that can affect different brain areas and induce significant neuronal damage [125]. In a study conducted on mouse brain endothelial cells, no positive effects were found following the administration of Andro, as the compound not only failed to improve the viability of these cells, but also induced a marked reduction in the number of neuronal cells due to increased LDH release and increased apoptosis, blocking cell growth in the G0/G1 phase [126]. However, from many other studies, especially those conduced in recent years, the protective action of Andro on ischemic damage seems to emerge and to be confirmed. More specifically, in a rat-mouse model with permanent middle cerebral artery occlusion (pMCAO), Andro reduced the infarct volume and microglia activation in peri-infarct areas by combatting increased levels of inflammatory markers such as IL-1β, TNF-α, and PGE2 and the transcription factor NFkB [127]. In another study, however, rats undergoing ischemic brain injury/reperfusion (CI/R) treated with Andro showed decreased cerebral infarction, reduced superoxide anion and nitrotyrosine, reduced expression of gp91phox/NOX2, IL-1β and hypoxia inducible factor (HIF), as well as reduced NFkB p65, thus confirming the protective effects of Andro in counteracting an ischemic attack [73]. Mice with CI/R-induced oxidative brain damage and treated with Andro also reported a reduced magnitude of infarction and subsequent neurological deficits, accompanied by reduced free radical production, as well as reduced nitrotyrosine, CD11b, NOX2, and iNOS formation [128].
Another andrographolide derivative, triacetylandrographolide (CX-10), has been used to test for possible neuroprotective effects against cerebral ischemia. On the macrophage cell line RAW264.7, both Andro and CX-10 counteracted NO and TNF-α production, while in BALB rats, CX-10 reduced LPS-induced TNF-α production. In Sprague Dawley rats with middle cerebral artery occlusion (MCAO), reduced infarct size and improved motor performance were observed. At the level of brain tissues, CX-10 reduced the levels of TNF-α and IL-1β and increased the activity of antioxidant enzymes such as SOD, CAT, and GSH-P. Western blot analysis also showed the positive effects of Andro in reducing the expression levels of TLR4, NF-κB, TNF-α, and iNOS proteins by increasing the expression of nuclear factor erythroid 2-related factor 2 (NRF2) and HO-1 [128]. In mouse cerebral endothelial cells (CECs) treated with 10µM ANDRO, an increase in HO-1 expression mediated by the phosphorylation of ERK1/2, p38 MAPK, and JNK1/2, and an increase was observed in Heme oxygenase 2 (HO-2) protein, via the NRF2 signaling pathway. In this way, Andro protected neuronal cells from cell death induced by oxygen-glucose deprivation (OGD), thus improving cell viability, while in rats subjected to MCAO, Andro's antioxidant effect in counteracting free radical production was observed, and a reduction in cerebral edema and infarct volume was detected in brain tissue analyses [129] (Figure 4). Sprague Dawley rats with middle cerebral artery occlusion (MCAO), reduced infarct size and improved motor performance were observed. At the level of brain tissues, CX-10 reduced the levels of TNF-α and IL-1β and increased the activity of antioxidant enzymes such as SOD, CAT, and GSH-P. Western blot analysis also showed the positive effects of Andro in reducing the expression levels of TLR4, NF-κB, TNF-α, and iNOS proteins by increasing the expression of nuclear factor erythroid 2-related factor 2 (NRF2) and HO-1 [128]. In mouse cerebral endothelial cells (CECs) treated with 10µM ANDRO, an increase in HO-1 expression mediated by the phosphorylation of ERK1/2, p38 MAPK, and JNK1/2, and an increase was observed in Heme oxygenase 2 (HO-2) protein, via the NRF2 signaling pathway. In this way, Andro protected neuronal cells from cell death induced by oxygen-glucose deprivation (OGD), thus improving cell viability, while in rats subjected to MCAO, Andro's antioxidant effect in counteracting free radical production was observed, and a reduction in cerebral edema and infarct volume was detected in brain tissue analyses [129] (Figure 4).

Conclusions
In conclusion, Ap and Andro exert neuroprotective effects on different NDs. The emerging information on the cytoprotective, anti-inflammatory, and antioxidant effects caused by the properties in Ap and Andro seems to explain many mechanisms at the heart of neuroprotective action. In particular, Andro can exert protective and therapeutic effects on certain central nervous system disorders, such as AD, PD, and cerebral ischemia, through its antioxidant and anti-inflammatory mechanisms. The neuroprotective effects of Andro may be mediated by several mechanisms, such as restoring blood-brain integrity, increasing synaptic plasticity, reducing the levels of specific chemokines and pro-inflammatory molecules activated during the neuroinflammation processes, promoting apoptosis and neurogenesis, and inhibiting Aβ aggregation. Indeed, both in vivo and in vitro studies have suggested that Andro shows considerable potential for development as a novel drug in the treatment of neurodegenerative diseases, and for this reason, it could also be considered as an adjuvant to conventional drugs therapies. Thus, a greater understanding of the neuroprotective molecular mechanisms of Ap and Andro could be crucial for identifying new therapeutic targets in NDs. However, in addition to its neuroprotective role, Ap and its major constituent Andro exhibit multiple pharmacological activities, including antioxidant, anti-inflammatory, anticancer,

Conclusions
In conclusion, Ap and Andro exert neuroprotective effects on different NDs. The emerging information on the cytoprotective, anti-inflammatory, and antioxidant effects caused by the properties in Ap and Andro seems to explain many mechanisms at the heart of neuroprotective action. In particular, Andro can exert protective and therapeutic effects on certain central nervous system disorders, such as AD, PD, and cerebral ischemia, through its antioxidant and anti-inflammatory mechanisms. The neuroprotective effects of Andro may be mediated by several mechanisms, such as restoring blood-brain integrity, increasing synaptic plasticity, reducing the levels of specific chemokines and pro-inflammatory molecules activated during the neuroinflammation processes, promoting apoptosis and neurogenesis, and inhibiting Aβ aggregation. Indeed, both in vivo and in vitro studies have suggested that Andro shows considerable potential for development as a novel drug in the treatment of neurodegenerative diseases, and for this reason, it could also be considered as an adjuvant to conventional drugs therapies. Thus, a greater understanding of the neuroprotective molecular mechanisms of Ap and Andro could be crucial for identifying new therapeutic targets in NDs. However, in addition to its neuroprotective role, Ap and its major constituent Andro exhibit multiple pharmacological activities, including antioxidant, anti-inflammatory, anticancer, antimicrobial, parasitic, hepatoprotective, antihyperglycemic, and antihyperglycemic actions, and experimental evidence has suggested that Andro may antagonize uncomplicated upper respiratory tract infections, including sinusitis and the common cold, as well as exert effects combatting rheumatoid arthritis. Furthermore, Andro does not appear to be particularly toxic; in fact, several studies have evaluated its efficacy and toxicity, even at high doses, demonstrating that it is a well-tolerated compound. However, some side effects have been documented, be problematic regarding the use of Andro, including gastrointestinal, skin, and subcutaneous tissue disorders, as well as anaphylaxis, general disorders and abnormal administration site conditions, and system disorders. Most of these adverse effects are mild to moderate, while a small number of patients may experience serious or life-threatening adverse effects. Most of these adverse reactions caused by Ap derivative injections can be relieved after drug discontinuation and symptomatic treatment [130]. Therefore, despite the fact that the use of Ap and Andro shows clear advantages, it is still necessary to carry out additional studies to improve the understanding of its therapeutic mechanisms and above all, to better understand and limit the disadvantages linked to its administration.

Conflicts of Interest:
The authors have no conflict of interest to declare. All co-authors have seen and agreed with the contents of the manuscript. We certify that this submission is an original work and is not under review at any other publication.