Andrographolide Inhibits Expression of NLPR3 Inflammasome in Canine Mononuclear Leukocytes.

Inflammasomes are multiprotein complexes that trigger processes through caspase-1 activation, leading to the maturation of proinflammatory cytokines, such as IL-1ß and IL-18. The gene encoding the inflammasome stimulatory protein NLRP3 is conserved in canines. Caspase-1/4 homologues have been identified in multiple carnivores, including canines, and caspase-1 activity has been shown in humans. The NLRP3 inflammasome has also been described in some canine inflammatory diseases. Andrographolide, a labdane diterpene, is the principal active ingredient in the herb Andrographis paniculate. The objective of this study was to determine the effect of andrographolide on the gene expression of the components of the NLRP3 inflammasome, proinflammatory cytokines, and IL-1ß secretion in canine peripheral blood mononuclear cells. For this, MTT assays and real-time PCR were employed to assess the cytotoxicity and gene expression. Further, an ELISA test was performed to measure the IL-1ß concentration. The findings reveal that andrographolide significantly reduces the expression of NLRP3, caspase-1/4, IL-1ß, and IL-18. Additionally, it decreases the secretion of IL-1ß and other proinflammatory cytokines, including IL-6, IL-8, and TNF-α. The results show that andrographolide decreases the expression of NLRP3, caspase-1/4, IL-1ß, and IL-18. Andrographolide also reduces proinflammatory cytokines expression, and decreases IL-1ß secretion. This indicates that andrographolide can interfere with the activation and function of the inflammasome, resulting in a decrease in the inflammatory response in canines. Research in this area is still budding, and more studies are necessary to fully understand andrographolide's mechanisms of action and its therapeutic potential in relation to the NLRP3 inflammasome in dogs.

Andrographolide Ameliorates Inflammatory Changes Induced by D-Lactate in Bovine Fibroblast-like Synoviocytes.

During acute ruminal acidosis, the manifestation of aseptic polysynovitis and lameness in cattle has been observed. Evidence suggests that joint inflammation can be attributed to the metabolic alterations induced by D-lactate in fibroblast-like synoviocytes (FLSs). We aimed to investigate whether andrographolide could mitigate the inflammation and metabolic alterations induced by D-lactate in bovine fibroblast-like synoviocytes (bFLSs). To assess this, bFLSs were cultured in the presence or absence of andrographolide. We evaluated its potential interference with the expression of proinflammatory cytokines, COX-2, HIF-1α, and LDHA using RT-qPCR. Furthermore, we investigated its potential interference with PI3K/Akt signaling and IκBα degradation through immunoblotting and flow cytometry, respectively. Our observations revealed that andrographolide reduced the elevation of IL-6, IL-8, COX-2, HIF-1α, and LDHA induced by D-lactate. Additionally, andrographolide demonstrated interference with the PI3K/Akt and NF-κB pathways in bFLSs. In conclusion, our findings suggest that andrographolide can potentially reverse the inflammatory effects and metabolic changes induced by D-lactate in bFLSs, showing promise as a therapeutic intervention for managing these conditions associated with lameness.

Age-Dependent Behavioral and Synaptic Dysfunction Impairment Are Improved with Long-Term Andrographolide Administration in Long-Lived Female Degus (Octodon degus).

In Octodon degus, the aging process is not equivalent between sexes and worsens for females. To determine the beginning of detrimental features in females and the ways in which to improve them, we compared adult females (36 months old) and aged females (72 months old) treated with Andrographolide (ANDRO), the primary ingredient in Andrographis paniculata. Our behavioral data demonstrated that age does not affect recognition memory and preference for novel experiences, but ANDRO increases these at both ages. Sociability was also not affected by age; however, social recognition and long-term memory were lower in the aged females than adults but were restored with ANDRO. The synaptic physiology data from brain slices showed that adults have more basal synaptic efficiency than aged degus; however, ANDRO reduced basal activity in adults, while it increased long-term potentiation (LTP). Instead, ANDRO increased the basal synaptic activity and LTP in aged females. Age-dependent changes were also observed in synaptic proteins, where aged females have higher synaptotagmin (SYT) and lower postsynaptic density protein-95 (PSD95) levels than adults. ANDRO increased the N-methyl D-aspartate receptor subtype 2B (NR2B) at both ages and the PSD95 and Homer1 only in the aged. Thus, females exposed to long-term ANDRO administration show improved complex behaviors related to age-detrimental effects, modulating mechanisms of synaptic transmission, and proteins.

Andrographolide Attenuates Short-Term Spatial and Recognition Memory Impairment and Neuroinflammation Induced by a Streptozotocin Rat Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder clinically manifested by a gradual cognitive decline. Intracerebroventricular injection (ICV) of streptozotocin (STZ), a model of sporadic AD (sAD), shows many aspects of sAD abnormalities (i.e., neuroinflammation, oxidative stress, protein aggregation), resulting in memory impairment. Andrographolide (ANDRO), a natural diterpene lactone, has numerous bioactivities including anti-inflammatory and antioxidant properties. Studies in rodents revealed that ANDRO has neuroprotective properties and restores cognitive impairment. In the present study, we investigated the effects of ANDRO in the ICV-STZ model relative to short-term spatial memory (object location test (OLT) and Y maze test), short-term recognition memory (object recognition test (ORT)), locomotor activity (open field test (OFT)), expression of amyloid precursor protein (APP), and activation of astrocytes (glial fibrillary acidic protein (GFAP) expression) and microglia (ionized calcium-binding adapter molecule-1 (Iba-1) immunohistochemistry) in the prefrontal cortex (PFC) and hippocampus (HIP). Wistar rats were injected ICV with STZ (3 mg/kg) or vehicle and treated with ANDRO (2 mg/kg, i.p.; three times per week). After four weeks, ANDRO attenuated the impairments of the Y maze and ORT performances, and the increase of astrocyte activation in the PFC induced by the ICV-STZ model. In addition, ANDRO decreased the number of activated microglia cells in the HIP of STZ-injected rats. The APP expression was not altered, neither by the STZ nor ANDRO. ANDRO showed a beneficial effect on memory impairment and neuroinflammation in the STZ model of AD.

Age- and Sex-Associated Glucose Metabolism Decline in a Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by a high etiological and clinical heterogeneity, which has obscured the diagnostic and treatment efficacy, as well as limited the development of potential drugs. Sex differences are among the risk factors that contribute to the variability of disease manifestation. Unlike men, women are at greater risk of developing AD and suffer from higher cognitive deterioration, together with important changes in pathological features. Alterations in glucose metabolism are emerging as a key player in the pathogenesis of AD, which appear even decades before the presence of clinical symptoms. OBJECTIVE: We aimed to study whether AD-related sex differences influence glucose metabolism. METHODS: We used male and female APPswe/PS1dE9 (APP/PS1) transgenic mice of different ages to examine glucose metabolism effects on AD development. RESULTS: Our analysis suggests an age-dependent decline of metabolic responses, cognitive functions, and brain energy homeostasis, together with an increase of Aß levels in both males and females APP/PS1 mice. The administration of Andrographolide (Andro), an anti-inflammatory and anti-diabetic compound, was able to restore several metabolic disturbances, including the glycolytic and the pentose phosphate pathway fluxes, ATP levels, AMPKα activity, and Glut3 expression in 8-month-old mice, independent of the sex, while rescuing these abnormalities only in older females. Similarly, Andro also prevented Aß accumulation and cognitive decline in all but old males. CONCLUSION: Our study provides insight into the heterogeneity of the disease and supports the use of Andro as a potential drug to promote personalized medicine in AD.

Effect of carrier materials on the properties of the andrographolide solid dispersion

Abstract In the work the andrographolide (AG)-solid dispersions (SDs) were prepared by the spray-drying method, using polyethylene glycol 8000 (PEG8000), Poloxamer188, polyvinylpyrrolidone K30 (PVPK30), Soluplus® as carrier materials. The effect of different polymers as carrier materials on the properties of the AG-SDs were studied. The results showed obvious differences in intermolecular interaction, thermal stability, drug state, powder properties, dissolution behavior, and so on of AG-SDs prepared using different polymers as carrier materials. AG-PEG8000-SD was a partial-crystalline and partial-amorphous powder with smaller surface area and pore volume, but it was easy to wetting and did not swell in contact with dissolved medium. AG-Soluplus®-SD was completely amorphous powder with larger specific surface area and pore volume, but it swelled in contact with water. Therefore, the dissolution profile of AG in AG-PEG8000-SD was similar to that in AG-Soluplus®-SD. Soluplus® and PEG8000 were suitable polymers to design AG-SDs, considering both physicochemical properties and dissolution behaviors. The results of this reseach showed that when selecting carrier materials for SD, we should not only consider the state of drugs in SD and the powder properties of SD, but also consider whether there is swelling when the carrier materials are in contact with the dissolution medium.

Andrographolide protects against isoproterenol-induced myocardial infarction in rats through inhibition of L-type Ca2+ and increase of cardiac transient outward K+ currents.

Myocardial infarction (MI) is the irreversible injury of the myocardium caused by prolonged myocardial ischemia and is a major cause of heart failure and eventual death among ischemic patients. The present study assessed the protective potentials of andrographolide against isoproterenol-induced myocardial infarction in rats. Animals were randomly divided into four groups: Control (Ctr) group received 0.9% saline solution once daily for 21 days, Isoproterenol (Iso) group received 0.9% saline solution once daily for 19 days followed by 80 mg/kg/day of isoproterenol hydrochloride solution on day 20 and 21, Andrographolide (Andro) group received 20 mg/kg/day of andrographolide for 21 days, and Andrographolide plus Isoproterenol (Andro + Iso) group received 20 mg/kg/day of andrographolide for 21 days with co-administration of 80 mg/kg/day of isoproterenol hydrochloride solution on day 20 and 21. After all treatments, cardiac-specific parameters that define cardiac health and early subacute MI were measured in all groups using both biophysical and pharmacological assay methods. Isoproterenol administration significantly (P < 0.05) increased cardiac mass indexes, systemic cardiac biomarkers, infarct size and caused cardiac histological alterations; significantly (P < 0.05) increased heart rate, QRS & QTc intervals and caused ST-segment elevation; significantly (P < 0.05) increased myocytes shortening, action potential duration (APD), L-type Ca2+ current (ICa,L) density and significantly (P < 0.05) decreased transient outward K+ current (Ito) density typical of the early subacute MI. Interestingly, pretreatment with andrographolide prevented and or minimized these anomalies, notably, by reducing ICa,L density and increasing Ito density significantly. Therefore, andrographolide could be seen as a promising therapeutic agent capable of making the heart resistant to early subacute infarction and it could be used as template for the development of semisynthetic drug(s) for cardiac protection against MI.

Andrographolide restores glucose uptake in rat hippocampal neurons.

Cerebral glucose hypometabolism is a common pathophysiological characteristic of many neurodegenerative diseases. This metabolic dysfunction includes alterations in glucose transport from the blood into the neurons by the facilitative glucose transporters (GLUTs). Several studies suggest that metabolic disturbances precede clinical symptoms and correlate with disease progression. Some groups have started to explore the use of therapeutic strategies that target decreased cerebral glucose metabolism to promote its availability. We selected Andrographolide (Andro), a natural product obtained from Andrographis paniculate that has both anti-hyperglycemic and anti-diabetic effects. Although it was shown to promote glucose uptake in vivo, the underlying mechanisms remain unclear. Here, we evaluated the acute effects of Andro on glucose transport and metabolism using primary rat hippocampal neuronal cultures. Our results showed that Andro enhances neuronal glucose uptake and stimulates glucose metabolism by inducing GLUT3 and 4 expression in neurons, as well as by promoting glycolysis. We also observed that Andro-mediated effects depend on the activity of AMP-activated protein kinase (AMPK), one of the central regulators of glucose metabolism. Our studies open the possibility to use Andro as a drug to restore glucose levels in neurodegenerative diseases.

Andrographolide, an Anti-Inflammatory Multitarget Drug: All Roads Lead to Cellular Metabolism.

Andrographolide is a labdane diterpene and the main active ingredient isolated from the herb Andrographis paniculata. Andrographolide possesses diverse biological effects including anti-inflammatory, antioxidant, and antineoplastic properties. Clinical studies have demonstrated that andrographolide could be useful in therapy for a wide range of diseases such as osteoarthritis, upper respiratory diseases, and multiple sclerosis. Several targets are described for andrographolide, including the interference of transcription factors NF-κB, AP-1, and HIF-1 and signaling pathways such as PI3K/Akt, MAPK, and JAK/STAT. In addition, an increase in the Nrf2 (nuclear factor erythroid 2-related factor 2) signaling pathway also supports its antioxidant and anti-inflammatory properties. However, this scenario could be more complex since recent evidence suggests that andrographolide targets can modulate glucose metabolism. The metabolic effect of andrographolide might be the key to explaining the diverse therapeutic effects described in preclinical and clinical studies. This review discusses some of the most recent evidence about the anti-inflammatory and metabolic effects of andrographolide.

Efficacy of andrographolide in not active progressive multiple sclerosis: a prospective exploratory double-blind, parallel-group, randomized, placebo-controlled trial.

BACKGROUND: Multiple sclerosis (MS) is a chronic immune mediated disease and the progressive phase appears to have significant neurodegenerative mechanisms. The classification of the course of progressive MS (PMS) has been re-organized into categories of active vs. not active inflammatory disease and the presence vs. absence of gradual disease progression. Clinical trial experience to date in PMS with anti-inflammatory medications has shown limited effect. Andrographolide is a new class of anti-inflammatory agent, that has been proposed as a potential drug for autoimmune disorders, including MS. In the present trial, we perform an exploratory pilot study on the efficacy and safety of andrographolide (AP) compared to placebo in not active PMS. METHODS: A pilot clinical trial using 140 mg oral AP or placebo twice daily for 24 months in patients with not active primary or secondary progressive MS was conducted. The primary efficacy endpoint was the mean percentage brain volume change (mPBVC). Secondary efficacy endpoints included 3-month confirmed disability progression (3-CDP) and mean EDSS change. RESULTS: Forty-four patients were randomized: 23 were assigned to the AP group, and 21 were assigned to the placebo group. The median baseline EDSS of both groups was 6.0. Annualized mPBVC was - 0.679% for the AP group and - 1.069% for the placebo group (mean difference: -0.39; 95% CI [- 0.836-0.055], p = 0.08, relative reduction: 36.5%). In the AP group, 30% had 3-CDP compared to 41% in the placebo group (HR: 0.596; 95% CI [0.200-1.777], p = 0.06). The mean EDSS change was - 0.025 in the AP group and + 0.352 in the placebo group (mean difference: 0.63, p = 0.042). Adverse events related to AP were mild rash and dysgeusia. CONCLUSIONS: AP was well tolerated and showed a potential effect in reducing brain atrophy and disability progression, that need to be further evaluated in a larger clinical trial. TRIAL REGISTRATION: ClinicalTrials.gov NCT02273635 retrospectively registered on October 24th, 2014.

Andrographolide Reduces Neuroinflammation and Oxidative Stress in Aged Octodon degus.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder in which superior brain functions, such as memory and cognition, are impaired. Currently, no effective treatment is available for AD. Although andrographolide (ANDRO), a compound extracted from the herb Andrographis paniculata, has shown interesting effects in models of several diseases, including AD, its effects on other molecular changes observed in AD, such as neuroinflammation and oxidative stress, have not yet been studied. To evaluate the impact of ANDRO-based intervention on the levels of amyloid-ß (Aß) and neuroinflammatory and oxidative stress markers in the brains of aged Octodon degus, a Chilean rodent, fifty-six-month-old O. degus were treated intraperitoneally with 2 or 4 mg/kg ANDRO. Vehicle-injected and 12-month-old O. degus were used as positive controls. Then, the protein levels of selected markers were assessed via immunohistochemistry and immunoblotting. ANDRO significantly reduced the total Aß burden as well as astrogliosis and interleukin-6 levels. Moreover, ANDRO significantly reduced the levels of 4-hydroxynonenal and N-tyrosine adducts, suggesting a relevant reduction in oxidative stress within aged O. degus brain. Considering that O. degus has been proposed as a potential "natural" model for sporadic AD due to the development of neuropathological markers that resemble this pathology, our results suggest that ANDRO should be further studied to establish its potential as a therapeutic drug for AD.

Presymptomatic Treatment With Andrographolide Improves Brain Metabolic Markers and Cognitive Behavior in a Model of Early-Onset Alzheimer's Disease.

Alzheimer's disease (AD) is the most common type of dementia. The onset and progression of this pathology are correlated with several changes in the brain, including the formation of extracellular aggregates of amyloid-beta (Aß) peptide and the intracellular accumulation of hyperphosphorylated tau protein. In addition, dysregulated neuronal plasticity, synapse loss, and a reduction in cellular energy metabolism have also been described. Canonical Wnt signaling has also been shown to be downregulated in AD. Remarkably, we showed previously that the in vivo inhibition of Wnt signaling accelerates the appearance of AD markers in transgenic (Tg) and wild-type (WT) mice. Additionally, we found that Wnt signaling stimulates energy metabolism, which is critical for the ability of Wnt to promote the recovery of cognitive function in AD. Therefore, we hypothesized that activation of canonical Wnt signaling in a presymptomatic transgenic animal model of AD would improve some symptoms. To explore the latter, we used a transgenic mouse model (J20 Tg) with mild AD phenotype expression (high levels of amyloid aggregates) and studied the effect of andrographolide (ANDRO), an activator of canonical Wnt signaling. We found that presymptomatic administration of ANDRO in J20 Tg mice prevented the reduction in cellular energy metabolism markers. Moreover, treated animals showed improvement in cognitive performance. At the synaptic level, J20 Tg animals showed severe deficiencies in presynaptic function as determined by electrophysiological parameters, all of which were completely restored to normal by ANDRO administration. Finally, an analysis of hippocampal synaptosomes by electron microscopy revealed that the length of synapses was restored with ANDRO treatment. Altogether, these data support the idea that the activation of canonical Wnt signaling during presymptomatic stages could represent an interesting pharmacological strategy to delay the onset of AD.

Andrographolide protects mouse astrocytes against hypoxia injury by promoting autophagy and S100B expression

Andrographolide (ANDRO) has been studied for its immunomodulation, anti-inflammatory, and neuroprotection effects. Because brain hypoxia is the most common factor of secondary brain injury after traumatic brain injury, we studied the role and possible mechanism of ANDRO in this process using hypoxia-injured astrocytes. Mouse cortical astrocytes C8-D1A (astrocyte type I clone from C57/BL6 strains) were subjected to 3 and 21% of O2 for various times (0-12 h) to establish an astrocyte hypoxia injury model in vitro. After hypoxia and ANDRO administration, the changes in cell viability and apoptosis were assessed using CCK-8 and flow cytometry. Expression changes in apoptosis-related proteins, autophagy-related proteins, main factors of JNK pathway, ATG5, and S100B were determined by western blot. Hypoxia remarkably damaged C8-D1A cells evidenced by reduction of cell viability and induction of apoptosis. Hypoxia also induced autophagy and overproduction of S100B. ANDRO reduced cell apoptosis and promoted cell autophagy and S100B expression. After ANDRO administration, autophagy-related proteins, S-100B, JNK pathway proteins, and ATG5 were all upregulated, while autophagy-related proteins and s100b were downregulated when the jnk pathway was inhibited or ATG5 was knocked down. ANDRO conferred a survival advantage to hypoxia-injured astrocytes by reducing cell apoptosis and promoting autophagy and s100b expression. Furthermore, the promotion of autophagy and s100b expression by ANDRO was via activation of jnk pathway and regulation of ATG5.