The antidepressant efficacy exhibits significant individual variation influenced by genetic factors. Traditional Chinese Medicine (TCM) as a personalized medicine is intrinsically contributed by genetic variation, but experimental investigation remains scarce. This study investigated the genetic impacts of the antidepressant effects of Yueju Pill (YJ), a classic TCM formula, using Balb/c and C57BL/6 J mouse strains, and the underlying electrophysiological and molecular mechanisms. We found that the same dose of YJ induced immediate and sustained antidepressant-like effects exclusively in Balb/c mice, accompanied by a specific enhancement of long-term potentiation (LTP) in the hippocampus. These effects were absent in C57BL/6 J mice. Further pharmacological validation confirmed the key role of the PKA-CREB pathway: treatment with a PKA signaling inhibitor H-89, administered either before or after YJ, completely blocked its antidepressant effects, indicating that this pathway is essential for both the initiation and maintenance of its efficacy. Notably, a comparative analysis with ketamine revealed mechanistic differences between the two: PKA activation was not required for the initiation of ketamine's antidepressant effect, and PKA signaling was only responsible for the early but short maintenance stage of ketamine's antidepressant effects. Furthermore, in the LPS-induced inflammatory depression model using Balb/c mice, YJ demonstrated antidepressant effects 24 h after a single administration and western blot analysis of hippocampal tissue revealed that YJ treatment significantly down‑regulated the protein expression of phospho‑NF‑κB (p‑NF‑κB) and the microglial marker Iba1, indicating a suppression of neuroinflammatory activation. In summary, differences in genetic background influenced the behavioral and electrophysiological effects of YJ. The activation of PKA signaling is required for both the initiation and early maintenance of its antidepressant actions, and attenuation of neuroinflammation may also contribute to the maintainence of its antidepressant effects.

BACKGROUND:The global rise in obesity and metabolic syndrome is increasingly recognized as a major risk factor for cognitive decline and neurodegenerative diseases. Chronic high-fat diet (HFD) consumption disrupts blood-brain barrier (BBB) integrity, a pivotal pathological event that facilitates neurotoxic infiltration, neuroinflammation, and neuronal injury. Brain pericytes play a central role in maintaining BBB function and are particularly vulnerable to HFD-induced metabolic stress. Loss or dysfunction of pericytes contributes to BBB breakdown. Low-density lipoprotein receptor-related protein 1 (LRP1), which is highly expressed in pericytes, is a critical regulator of neurovascular integrity; its downregulation activates the CypA/NF-κB/MMP-9 signaling cascade, thereby exacerbating BBB permeability. PURPOSE:This study aimed to investigate whether the traditional Chinese medicine formula Zexieyin Formula (ZXYF) ameliorates HFD-induced cognitive impairment by targeting pericyte dysfunction. We hypothesized that ZXYF exerts neuroprotective effects by restoring pericyte-associated LRP1 expression, suppressing activation of the CypA/NF-κB/MMP-9 pathway, preserving BBB integrity, and consequently attenuating hippocampal neuronal damage. Both in vivo and in vitro models were employed to elucidate the underlying neurovascular mechanisms. METHODS:In vivo, C57BL/6 mice were fed an HFD for 16 weeks to induce cognitive impairment, followed by a 4-week intervention with ZXYF; atorvastatin (ATO) served as a positive control. Cognitive function was evaluated using a battery of behavioral tests. BBB integrity was assessed by transmission electron microscopy (TEM) and Western blot analysis of the tight junction proteins ZO-1 and Claudin-5. Cerebrovascular permeability was further evaluated using sodium fluorescein extravasation assays combined with co-localization analysis with the endothelial marker CD31. Pericyte-associated LRP1 expression in the hippocampus was examined by immunofluorescent co-localization of LRP1 with the pericyte marker PDGFR-β. Activation of the CypA/NF-κB/MMP-9 signaling pathway in hippocampal tissue was analyzed by Western blotting. In vitro, mouse brain microvascular pericytes (MBVPs) were exposed to free fatty acids (FFA) to model metabolic stress. LRP1 expression and localization were assessed by confocal microscopy, and protein levels of the CypA/NF-κB/MMP-9 pathway were determined by Western blotting. An LRP1-knockdown MBVPs cell line was generated via lentiviral transduction to evaluate LRP1 dependence. RESULTS:HFD-fed mice exhibited pronounced cognitive deficits, which were significantly ameliorated by ZXYF treatment. TEM and Western blot analyses revealed marked BBB disruption in HFD-fed mice, characterized by ultrastructural abnormalities and reduced expression of ZO-1 and Claudin-5. Immunofluorescence demonstrated increased sodium fluorescein leakage in the hippocampus, indicating cerebrovascular damage, which was partially reversed by ZXYF. Mechanistically, confocal imaging showed a significant reduction in LRP1 expression in hippocampal pericytes of HFD-fed mice, accompanied by activation of the CypA/NF-κB/MMP-9 pathway. ZXYF intervention restored pericyte-associated LRP1 expression and suppressed pathway activation. Consistently, lentivirus-mediated LRP1 knockdown in MBVPs abolished the inhibitory effects of ZXYF on CypA/NF-κB/MMP-9 signaling, demonstrating that ZXYF-mediated pathway regulation is LRP1-dependent. CONCLUSION:This study demonstrates that ZXYF alleviates HFD-induced cognitive impairment by targeting BBB function. ZXYF restores pericyte-associated LRP1 expression, thereby inhibiting the CypA/NF-κB/MMP-9 signaling pathway, preserving BBB integrity, and protecting hippocampal neurons. These findings identify pericytes and the LRP1/CypA/NF-κB/MMP-9 axis as critical therapeutic targets in metabolic cognitive disorders and provide a novel mechanistic basis for the neuroprotective effects of traditional Chinese medicine.

BackgroundThe hemoglobin glycation index (HGI) reflects discordance between measured HbA1c and glucose-predicted HbA1c, but in acute ischemic stroke (IS) it may also capture stress-hyperglycemia-related fasting plasma glucose (FPG) elevation. We examined the association of HGI with large-artery atherosclerosis (LAA) and in-hospital mortality.MethodsWe retrospectively analyzed 4,500 IS patients from MIMIC-IV and 330 patients from an external clinical cohort. HGI was calculated using cohort-specific FPG-HbA1c regression equations. Multivariable logistic regression and restricted cubic spline analyses assessed associations with LAA and mortality. Boruta feature selection, exploratory Mendelian randomization (MR), and post hoc stress hyperglycemia ratio (SHR) sensitivity analyses were also performed.ResultsLower HGI was independently associated with higher LAA risk in both cohorts (MIMIC-IV: OR = 0.579, p < 0.001; clinical cohort: OR = 0.599, p < 0.001), with an L-shaped nonlinear pattern. In MIMIC-IV, lower HGI was also associated with higher in-hospital mortality (OR = 0.488, p < 0.001). HGI showed the highest Boruta feature importance among measured baseline variables. HGI and SHR were strongly inversely correlated in both cohorts (Spearman’s ρ = −0.711 and −0.723; both p < 0.001), and higher SHR showed directionally consistent associations with adverse outcomes. MR did not provide significant genetic evidence linking HGI to LAA.ConclusionLow HGI was associated with higher LAA risk and in-hospital mortality in IS. This signal overlaps with stress-hyperglycemia-related FPG-HbA1c discordance; therefore, HGI may be useful for risk stratification but should not be interpreted as an independent causal glycation phenotype.

Myasthenia gravis (MG) is an autoimmune di sease characterized by muscle weakness. Experimental autoimmune myasthenia gravis (EAMG) serves as an animal model for MG research. Despite advancements in EAMG modeling, limited drug absorption and variability in disease manifestation among animals resulted in a low success rate of model induction. This study aimed to optimize and standardize the modeling process by exploring different induction conditions to improve success rates. We employed female Lewis rats and C57BL/6 mice to compare their sensitivity to model induction and applied a controlled variable approach to acetylcholine receptor (AChR) and H37Ra dosage, mixing time, and injection sites. Results showed that Lewis rats were more suitable than C57BL/6 mice, and 75 µg AChR peptides were more effective than 50 µg. The immune-boosting effect of 1 mg H37Ra Mycobacterium tuberculosis was weaker than 2 mg. While drug mixing time had little impact, increasing injection sites on backs and including foot pads injection, significantly improved drug absorption.

The chronic inflammatory state associated with vascular aging plays a critical role in the pathogenesis of age-related diseases, including occurrence and progression of atherosclerosis (AS) and cognitive impairment (CI). The detrimental impacts of inflammation are multifaceted. On the one hand, inflammation impairs the function of vascular endothelial cells (EC) and contributes to vascular remodeling. On the other hand, it triggers a cascade of events resulting in cerebral small vessel disease (CSVD), which disrupts the blood-brain barrier (BBB), induces neuroinflammation and ultimately impairs cognitive function. Consequently, these processes establish a strong pathological link between atherosclerosis (AS) and cognitive impairment (CI). Chinese Herbal Medicine(CHM) posits that AS and CI share common pathological factors and similar pathogeneses. According to CHM theory, the aging process is associated with a gradual deficiency of healthy qi, which subsequently results in spleen qi deficiency and impaired transportation functions. This dysfunction leads to the accumulation of water-dampness, which can further transform into pathogenic fire, thereby causing damage to the veins and channels. Clinically, such pathological changes may manifest as cardiac pain, dementia, and other symptoms. The treatment of AS in CHM follows principles such as clearing heat, removing toxins, as well as eliminating phlegm and resolving blood stasis. It is notable that numerous studies have demonstrated that Chinese herbs exhibit distinctive therapeutic potential in modulating the progression of CI associated with AS. These herbs have been observed to target multiple pathways intricately involved in AS-related inflammatory processes and cognitive impairment, offering a comprehensive therapeutic approach. In light of the above, this review aims to provide an overview of the evidence supporting the association between AS and CI. It will explore the pathological mechanisms and processes by which AS contributes to the development of CI, and summarize the mechanism of action of Chinese herbs in the treatment of AS-related CI. The findings aim to inform future therapeutic strategies for cognitive disorders and guide research directions in this field.

Atherosclerosis (AS) is the most common cardiovascular disease (CVD), despite an overall declining incidence, AS remains a leading cause of death worldwide. The ZeXieYin formula (ZXYF), one of the thirteen formulas recorded in HuangDiNeiJin, a classical book of Traditional Chinese Medicine (TCM), has previously demonstrated efficacy in reducing blood lipids and combating AS. However, the precise mechanism by which it regulates blood lipids remains unclear. Given the close correlation between bile acid metabolism and cholesterol metabolism, it is imperative to elucidate the intrinsic mechanisms through which ZXYF treats AS. This study aims to investigate the pivotal role of enterohepatic bile acid circulation in enhancing intestinal barrier function and mitigating AS by ZXYF. The AS model was established by subjecting male ApoE−/− mice to a high-fat diet (HFD). Moreover, to determine the impact of ZXYF on the integrity of the intestinal barrier, we quantified proinflammatory cytokines using RT-qPCR and ELISA. Additionally, we identified tight-junction proteins in the ileal tissues through IF. Finally, the intestinal flora metabolite and fecal bile acid composition were analyzed using 16S rRNA analysis, untargeted metabolomics analysis, and targeted metabolomics analysis. The ZXYF significantly improved dyslipidemia and alleviated the formation of arterial plaques in AS mice. Furthermore, the administration of ZXYF resulted in a concurrent reduction in circulating lipopolysaccharide (LPS) levels and downregulation of pro-inflammatory cytokine mRNA expression in the ileum. Additionally, there was an enhancement observed in the expression of tight junction proteins within the intestinal tissue of AS mice. Further studies found that ZXYF significantly elevated the total bile acids (TBA) and total cholesterol (TC) levels in the fecal of AS mice. The untargeted and targeted metabolomic analyses further revealed that ZXYF exerts regulatory effects on bile acid phenotype by decreasing secondary bile acids (SBAs) levels through modulation of gut microbiota composition, such as enrichment of Akkermansia (AKK) abundance, and inhibition of enterohepatic circulation of bile acids. ZXYF specifically increased the expression of hepatic bile acid synthesis enzymes CYP7A1 by modulating the FXR/FGF15 signaling pathway, thereby promoting enhanced de novo bile acid synthesis and facilitating cholesterol catabolic excretion. The findings of our research indicate that ZXYF exerts a defensive role in the advancement of AS. The mechanism underlying the role of ZXYF in combating AS is closely associated with gut microbiota reshaping and regulation of enterohepatic bile acid circulation.

Buyang Huanwu Dcoction (BYHWD) is a traditional Chinese medicine that has been widely used for the clinical treatment of skeletal muscle atrophy which is a common complication after motor neuron injury and seriously affects the recovery of skeletal muscle function. This study aimed to explore the main components of Buyang Huanwu decoction and its possible mechanism for treating skeletal muscle and nerve atrophy in mice. Total fibular nerve injury model was established by using total fibular nerve clamp surgery. Main component of decoction was detected by HPLC and LC-MS, the morphology of skeletal muscle samples was observed using hematoxylin-eosin staining (H&E) and laser lens, and the expressions of muscular atrophy related proteins were detected by Western blot. Astragaloside is the main components of BYHWD, it promotes the recovery of injured neuroskeletal muscle and significantly inhibits the atrophy of related muscle tissue (P<0.05). Denervation of skeletal muscle is closely related to autophagy, and Astragaloside can effectively inhibit autophagy after skeletal muscle injury. Astragaloside, the main component of BYHWD, promotes the recovery of skeletal muscle denervation by inhibiting autophagy.

Background:Parkinson's disease (PD) is a complex neurodegenerative disease, which is often treated with obvious side effects such as dopamine replacement therapy. Our team has validated the unique advantages of the traditional Chinese medicine formula, YiQiWenYangSanHan formula (YQWYSHF), through in vitro experiments, confirming its therapeutic potential for PD. Nevertheless, further research and validation are required to fully understand its protective effects and underlying mechanisms against PD. Aim of this review:This study employed an in vivo model to investigate the effects of YQWYSHF on motor impairments, neuroinflammation, and mitochondrial dysfunction in C57BL/6 J mice caused by MPTP. Materials and methods:Sixty C57BL/6 J mice were randomly divided into 5 groups, all groups except the control group were intraperitoneally administered MPTP for 7 days (30 mg/kg). After 4 weeks of drug intragastric treatment, we assessed the dyskinesia of mice treated with different doses of YQWYSHF by behavioral examination. Additionally, immunofluorescence was used to examine the expression of ionized calcium binding adaptor protein 1 (IBA1) and glial fibrillary acidic protein-positive (GFAP) cells. Western blotting was used to assess the expression level of tyrosine hydroxylase (TH), pyrin domain-containing 3 protein (NLRP3), apoptosis-associated speck-like proteins (ASC), cysteine-containing aspartate protease-1 (Caspase-1), interleukin-1β (IL-1β), α-synuclein (α-syn), poly (ADP-ribose) polymerase 1 (PARP1), and poly ADP ribose (PAR). Furthermore, transmission electron microscopy revealed mitochondrial impairment in the neuronal cells of the substantia nigra (SN). Results:YQWYSHF treatment alleviated dyskinesia in a mouse model of PD. Moreover, it increased the TH expression, and could reverse the increase of IBA1, GFAP, NLRP3, ASC, caspase-1,IL-1β, α-syn, PARP1 and PAR proteins induced by MPTP. Conclusions:YQWYSHF protects dopaminergic neurons in PD by attenuating neuroinflammation and mitochondrial dysfunction. This study provides new evidence for the clinical application of traditional Chinese medicine in the treatment of PD.

Atherosclerosis-related vascular cognitive impairment (VCI) is associated with dysregulated cholesterol metabolism and impaired autophagy. Alisol A, a natural tetracyclic triterpenoid derived from the traditional ZeXieYin Formula, has demonstrated anti-atherosclerotic and neuroprotective effects. However, its role in modulating brain cholesterol homeostasis and mitophagy in VCI remains largely unexplored. Methods: To elucidate the mechanism of Alisol A and evaluate its translational relevance, we employed an Ldlr-/- mouse model of VCI induced by a high-fat diet and left common carotid artery ligation. Alisol A was administered intragastrically, and cognitive function was assessed using the Morris water maze, Y-maze, and novel object recognition tests. To probe the role of NAMPT, pharmacological inhibition and lentiviral overexpression strategies were applied. Mechanistic investigations included Western blotting, immunofluorescence, and transmission electron microscopy to examine cholesterol metabolism, oxidative stress, mitophagy, and synaptic plasticity. Additionally, molecular docking, surface plasmon resonance, and lipidomic profiling were used to explore Alisol A-NAMPT binding and downstream regulatory pathways. Results: Alisol A significantly ameliorated cognitive impairment in Ldlr-/- mice. Mechanistically, it restored cholesterol homeostasis by activating the AMPK/NAMPT/SIRT1 signaling axis, upregulated UCP2 to suppress oxidative stress, and inhibited glial activation, thereby preserving neuronal structure and function. Additionally, Alisol A reactivated mitophagic flux by enhancing PINK1/PARKIN signaling and facilitating the clearance of damaged mitochondria, ultimately improving mitochondrial function. NAMPT was identified as a key molecular target mediating these neuroprotective effects. Conclusion: Alisol A confers neuroprotection in VCI by regulating cholesterol metabolism, attenuating oxidative stress, and restoring mitophagy via NAMPT-mediated signaling. These findings highlight its therapeutic potential in atherosclerosis-related cognitive decline.

Major depressive disorder (MDD) is a debilitating psychiatric condition associated with substantial personal, societal, and economic costs. Despite considerable advances in research, most conventional antidepressant therapies fail to achieve adequate response in a significant proportion of patients, underscoring the need for novel, mechanism-based interventions. Lycium barbarum glycopeptide (LbGp), a bioactive compound with emerging neuroprotective properties, has been proposed as a candidate for antidepressant development; however, its therapeutic efficacy and underlying mechanisms remain largely uncharacterized. In this study, ventral forebrain organoids were generated from patients with MDD to investigate disease-related neurophysiological abnormalities. These organoids exhibited disrupted neuronal morphology, diminished calcium signaling, and impaired electrophysiological activity. Administration of LbGp effectively restored structural and functional deficits in MDD-derived organoids. Transcriptomic profiling revealed that LbGp ameliorated endoplasmic reticulum (ER) stress responses. To investigate the causative role of ER stress, control organoids were treated with the ER stress agonist CCT020312, which elicited neural activity impairments resembling those observed in MDD organoids. Notably, LbGp reversed the phenotypic consequences of CCT020312 exposure in control organoids. In conclusion, ventral forebrain organoids derived from individuals with MDD demonstrated that LbGp ameliorates disease-associated phenotypes by modulating ER stress.

ETHNOPHARMACOLOGICAL RELEVANCE:Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies, the pathogenesis of which is well defined but for which safe and effective drugs are still lacking. Shengxian Decoction (SXD) is a classical Chinese herbal formula that has been shown to improve qi deficiency and fatigue syndrome, which is also one of the key pathomechanisms in myasthenia gravis. However, the potential of this formula to treat myasthenia gravis (MG) and its underlying mechanisms remains to be fully elucidated. AIM OF THE STUDY:To investigate the therapeutic effect of the traditional Chinese medicine formula, Shengxian Decoction (SXD) on MG and its potential mechanism of action. METHODS AND MATERIALS:Employing prednisone (PDN) as a positive control, the experimental autoimmune myasthenia gravis (EAMG) rat model induced by recombinant acetylcholine receptor (AChR) α subunit 97-116 peptide was used to reveal and confirm the therapeutic effect of SXD on MG. Model evaluation was performed based on animal behavioral performance combined with repeated nerve stimulation (RNS) experiments. Hematoxylin-Eosin (HE) staining and enzyme-linked immunosorbent (ELISA) assay were used to detect the level of inflammatory cytokines. The alterations in T-cell immunity were observed using flow cytometry (FCM). Immunohistochemistry (IHC) and immunofluorescence (IF) experiments were used to detect the molecular expression of TAZ, TEAD, and FOXP3. The ex vivo and in vivo mechanisms were validated by Western blot (WB) and Co-immunoprecipitation (Co-IP). RESULTS:The results showed that SXD significantly reversed loss of weight and muscle strength in EAMG rats, reducing the inflammation level and repairing the damage of thymic tissues in the model rats. Furthermore, SXD altered the trend observed in EAMG rats by markedly increasing the proportion of pro-inflammatory Th1 and Th17 cells and reducing the expression of Regulatory T cells (Treg), which plays a pivotal role in regulating the immune response in EAMG rats. SXD was shown to upregulate the expression of FOXP3 and TEAD, downregulate the expression of TAZ, and inhibit the binding of TAZ and TEAD in this research. This phenomenon was subsequently validated in vitro, which may elucidate the potential mechanism of SXD in the treatment of EAMG. CONCLUSION:SXD has been demonstrated to possess the capacity to treat EAMG. The underlying mechanism of action may be achieved by promoting the degradation of TAZ through the TAZ/TEAD pathway and inhibiting the binding of TAZ and TEAD, thereby restoring the balance of the body's immune function.

BACKGROUND:Disruption of the blood-brain barrier (BBB) is a critical pathological event in Alzheimer's disease (AD) progression. The ZeXieYin Formula (ZXYF), as described in the ancient Chinese medical text Huangdi Neijing, has shown multitarget neuroprotective effects and promising pharmacokinetics in preclinical studies with transgenic AD rodent models. Despite these findings, the exact molecular mechanisms by which its bioactive components interact with BBB regulatory pathways are not fully understood, necessitating comprehensive analysis through integrated systems pharmacology approaches. PURPOSE:The primary objective of this study is to explore the protective properties of ZXYF against BBB disruption in the context of AD. Additionally, the investigation seeks to elucidate the molecular pathways underlying the therapeutic effects of ZXYF in this scenario. METHODS:In vivo, APP/PS1 mice modeled AD. Y-maze and MWM tests assessed ZXYF's effects on cognition. Transmission electron microscopy (TEM) evaluated ZXYF's impact on BBB ultrastructure, while immunohistochemistry (IHC) and western blotting (WB) quantified tight junction (TJ) protein expression. Immunofluorescence detected GFAP (astrocytic marker), and ELISA measured hippocampal neuroinflammatory cytokines. Bioactive ZXYF components in systemic circulation were identified via UPLC-Q-TOF-MS/MS, followed by compound-target network construction and computational prioritization of AD pathways via multiplex network analysis. In vitro, ZXYF (2/6 mg/ml, 24 h) was applied to two BBB models: (1) LPS+TNF-α+IL-1α-stimulated bEnd.3 monolayers and (2) bEnd.3/C8-D1A astrocyte co-cultures. qPCR and WB assessed A1-specific astrocyte gene and protein expression. Molecular docking, Molecular dynamics (MD), Cellular thermal shift assay (CETSA) and Surface plasmon resonance (SPR) binding analysis simulations characterized ZXYF constituent binding to JAK2. Furthermore, siRNA was applied to knockdown JAK2 in the C8-D1A cell to further verify the role of JAK2/STAT3 pathway in ZXYF inhibition of A1 astrocyte activation. RESULTS:Our findings demonstrated that ZXYF preserved BBB integrity and improved cognitive function in AD mice. Mechanistically, ZXYF restored TJ protein expression (ZO-1, occludin, claudin-5), attenuated astrocyte activation (GFAP↓), and reduced neuroinflammation. Systemic component analysis identified 13 major bioactive constituents of ZXYF. Network pharmacology and GO/KEGG enrichment revealed the JAK2/STAT3 pathway as the core mechanism underlying ZXYF's anti-AD effects. In vitro, ZXYF protected endothelial cells in co-cultured BBB models against LPS/cytokine-induced injury by suppressing A1 astrocyte polarization. Crucially, molecular docking/dynamics confirmed strong binding affinity of key ZXYF components to JAK2, while WB validated ZXYF-mediated inhibition of JAK2/STAT3 phosphorylation (p-JAK2↓, p-STAT3↓) in AD mice. CONCLUSIONS:Our study shows that ZXYF significantly improved cognitive impairments and maintained BBB integrity in an AD mouse model. Compositional analysis revealed 13 major bioactive components of ZXYF. Mechanistically, ZXYF inhibited A1 astrocyte activation by suppressing the JAK2/STAT3 signaling pathway, enhancing endothelial barrier function. These results provide a mechanistic foundation for further investigation of ZXYF's therapeutic potential for AD and other cognitive disorders involving BBB dysfunction.

Ethnopharmacological relevanc: Pyrolae herba (PH), a kind of Chinese herb, has been identified to have an antiinflammatory effect, while the potential for treating cognitive impairment (CI), as well as the underlying mechanisms, is unclear. Currently, the interaction between neuroinflammation and neural function play a critical role in pathophysiology of CI.Aim of the study: To elucidate therapeutic effect of PH for CI as well as its underlying mechanisms with LPStreated mice model.Methods and materials: In this study, male C57BL6/J mice received lipopolysaccharide (LPS) injection for 10 days to establish CI model and were administrated with PH for 14 days. We used piracetam as a positive control. Memory and spatial function was tested by Morris water maze (MWM). The level of inflammation-related cytokines (TNF-alpha, IL-1 beta, IL-10, IL-6) were determined by enzyme-linked immunosorbent assay (ELISA) in serum and western blot in hippocampus. Immunofluorescence (IF) was used to measure the levels of ionized calcium binding linker molecule 1 (IBA-1), glial fibrillary acidic protein (GFAP), BrdU, Ki67 and doublecortin (DCX) in hippocampus. The mRNA sequencing was used to screen the potential target of PH with therapeutic CI. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the gene alteration of triggering receptor expressed on myeloid cells 2 (TREM2) in hippocampus. We used western blot to determine protein expressions of TREM2 and its related signaling, as well as synaptic proteins in hippocampus.Results: The results revealed that LPS contributed to CI, and PH or piracetam treatment significantly ameliorated CI in MWM test. LPS contributed to increasing expressions of TNF-alpha and IL-1 beta in serum and hippocampus, which both reversed by PH or piracetam. PH or piracetam could inhibit the activation of glial cells including microglia and astrocyte in the hippocampus in LPS-induced CI model. The mRNA sequencing and RT-PCR results showed that LPS significantly increased the gene expression of TREM2, which was reversed by PH. The alteration of TREM2 expression was the most significant among the 10 genes (TREM2, Slc24a2, Ptch2, Gck, Il1rapl1, Cadps2, Btbd11, Secisbp2l, Tenm3 and Prepl) in hippocampus. Protein results showed that LPS upregulated the expressions of TREM2 and its related proteins including DAP12, spleen tyrosine kinase (SYK) phosphorylation and ADAM 10, which were all reversed by PH or piracetam in hippocampus. Furthermore, LPS was capable of reducing the expression of BrdU and DCX co-labeled positive cells in hippocampal dentate gyrus (DG), which was reversed only by PH. Moreover, PH or piracetam treatment significantly increased the expression of Ki67 and DCX co-labeled positive cells in hippocampal DG. The expression of synapsin1 was obviously decreased by LPS , was significantly reversed by PH or piracetam.Conclusions: PH could alleviate CI by suppressing the secretion of pro-inflammatory cytokines and mitigating astrocyte activity by restraining microglia's activation in hippocampus, further facilitating neurogenesis and proliferation, thereby enhancing pre-synaptic protein. This study highlighted on the clinical application of PH, which might promote the use of phytomedicine in CI patients.

Ethnopharmacological relevance: Zexieyin formula (ZXYF), a traditional Chinese herbal formula recorded in the Huangdi Neijing to have efficacy in relieving spleen dampness and heat accumulation syndrome, which is also the key pathogenesis of atherosclerosis (AS). The efficacy has demonstrated by our previous studies. However, the intrinsic mechanism of ZXYF for treating vascular inflammation and the effect of inflammatory response on plaque are not known. Currently, plaque stabilization is crucial for the prognosis of AS. Aim of the study: Our study mainly focused on the therapeutic effects of ZXYF on high -fat diet (HFD)-induced vascular inflammation and vulnerable plaques (VP) in mice and explored its underlying mechanism. Methods and materials: Male apolipoprotein E knockout (APOE-/-) mice were fed HFD for 8 weeks to establish a VP model. During this period, the mice were also administered ZXYF, while atorvastatin (ATO) was used as a positive control. Aortic plaque area and morphology were detected by oil red staining and HE staining. Aortic plaque collagen content was detected by Masson staining. M1/M2 type macrophages were detected using immunofluorescence (IF). The study analyzed the levels of inflammation -related cytokines (IL -18, IL -10, IL -6), MAPK/NF-kappa B pathway proteins, and NLRP3 inflammasomes (NLRP3, Caspase-1) using Western blot. Additionally, the levels of matrix metalloproteinase (MMP)-2 and MMP-9 and alpha-smooth muscle actin (alpha-SMA) in the aorta were analyzed using immunohistochemistry (IHC). The plaque instability index was calculated for each group using the vulnerable plaque formula. Results: In this study, APOE-/- mice were fed high -fat diet for 8 weeks. The results of oil -red and HE staining indicated a significant increase in the aortic plaque area of the mice, which exhibited a typical VP phenotype. ZXYF and ATO significantly improved AS plaques and prevented plaque rupture. HFD exacerbated vascular inflammation, stimulated macrophage conversion to M1 -type through the MAPK/NF-kappa B signaling pathway, and released pro -inflammatory factors such as interleukin (IL) -18, IL-1 alpha, and IL -6. These factors activated NLRP3 inflammasome, leading to cellular death. However, ZXYF could reverse this trend and promote the conversion of macrophages to the anti-inflammatory M2 type. The anti-inflammatory effect of ATO was not significant. Moreover, HFD promoted the release of MMP-2 and MMP-9 from macrophages, which degraded plaque collagen, and induced a decrease in plaque SMC content, resulting in a thinning of the plaque fibrous cap. In contrast, ZXYF inhibited the decomposition of plaque collagen and increased the content of plaque smooth muscle cells (SMC) by reducing macrophage secretion of MMPs, thereby stabilizing plaques. Although ATO could reverse the decrease in plaque collagen and SMC content, its effect on MMPs was not significant. Finally, we calculated the vulnerability index to assess the overall risk of the plaque vulnerability phenotype. In line with these findings, ZXYF and ATO were able to effectively reverse the increase in the vulnerability index caused by HFD and lower the risk of adverse cardiovascular events. Conclusion: Our results suggested that ZXYF could reduce inflammation and increase plaque stability by inhibiting the MAPK/NF-kappa B signaling pathway, which provided a theoretical basis for clinical application and subsequent research.

ABSTRACT:Arteriosclerosis (AS) is a chronic inflammatory disease and Buyang Huanwu decoction (BHD) has been identified as an anti-atherosclerosis effect, and the study is aimed to investigate the underlying mechanism. The E4 allele of Apolipoprotein E (ApoE) is associated with both metabolic dysfunction and an enhanced pro-inflammatory response, ApoE-knockout (ApoE-/-) mice were fed with a high-fat diet to establish an arteriosclerosis model and treated with BHD or atorvastatin (as a positive control). The atherosclerotic plaque in each mouse was evaluated using Oil red O Staining. Elisa kits were used to evaluate blood lipid, interleukin-6 (IL-6), IL-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), IL-4, IL-10, and tumor growth factor beta (TGF-β) contents, while Western blot was applicated to measure inducible nitric oxide synthase (iNOS), arginase I (Arg-1) expression. Meanwhile, pyruvate kinase M2 (PKM2), hypoxia-inducible factor-1 alpha (HIF-1α) and its target genes glucose transporter type 1 (GLUT1), lactate dehydrogenase A (LDHA), and 3-phosphoinositide-dependent kinase 1 (PDK1), as well as IL-6, IL-1β, TNF-α, IL-4, IL-10, and TGF-β were evaluated by the quantitative reverse transcription-polymerase chain reaction. BHD treatment significantly reduced body weight and arteriosclerosis plaque area and blood lipid levels including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Meanwhile, BHD demonstrated a significant suppression of M1 polarization, by decreased secretion of iNOS and pro-inflammatory factors (IL-6, IL-1β, and TNF-α) in ApoE-/- mice. The present study also revealed that BHD promotes the activation of M2 polarization, characterized by the expression of Arg-1 and anti-inflammatory factors (IL-4 and IL-10). In addition, PKM2/HIF-1α signaling was improved by M1/M2 macrophages polarization induced by BHD. The downstream target genes (GLUT1, LDHA, and PDK1) expression was significantly increased in high fat feeding ApoE-/- mice, and those of which were recused by BHD and Atorvastatin. These results suggested that M1/M2 macrophages polarization produce the inflammatory response against AS progress after BHD exposure.

Background:The development of ketamine-like rapid antidepressants holds promise for enhancing the therapeutic efficacy of depression,but the underlying cellular and molecular mechanisms remain unclear.Implicated in depression regulation,the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is investigated here to examine its role in mediating the rapid antidepressant response.Methods:The onset of antidepressant response was assessed through depression-related behavioral paradigms.The signaling mechanism of PACAP in the hippocampal dentate gyrus (DG) was evaluated by utilizing site-directed gene knockdown,pharmacological interventions,or optogenetic manipulations.Overall,446 mice were used for behavioral and molecular signaling testing.Mice were divided into control or experimental groups randomly in each experiment,and the experimental manipulations included:chronic paroxetine treatments (4 d,9 d,14 d) or a single treatment of ketamine;social defeat or lipopolysaccharides-injection induced depression models;different doses of PACAP(0.4 ng/site,2 ng/site,4 ng/site;microinjected into the hippocampal DG);pharmacological intra-DG interventions(CALM and PACAP6-38);intra-DG viral-mediated PACAP RNAi;and opotogenetics using channelrhodopsins 2 (ChR2)or endoplasmic natronomonas halorhodopsine 3.0 (eNpHR3.0).Behavioral paradigms included novelty suppressed feeding test,tail suspension test,forced swimming test,and sucrose preference test.Western blotting,ELISA,or quantitative real-time PCR (RT-PCR) analysis were used to detect the expressions of proteins/peptides or genes in the hippocampus.Results:Chronic administration of the slow-onset antidepressant paroxetine resulted in an increase in hippocampal PACAP expression,and intra-DG blockade of PACAP attenuated the onset of the antidepressant response.The levels of hippocampal PACAP expression were reduced in both two distinct depression animal models and intra-DG knockdown of PACAP induced depression-like behaviors.Conversely,a single infusion of PACAP into the DG region produced a rapid and sustained antidepressant response in both normal and chronically stressed mice.Optogenetic intra-DG excitation of PACAP-expressing neurons instantly elicited antidepressant responses,while optogenetic inhibition induced depression-like behaviors.The longer optogenetic excitation/inhibition elicited the more sustained antidepressant/depression-like responses.Intra-DG PACAP infusion immediately facilitated the signaling for rapid antidepressant response by inhibiting calcium/calmodulin-dependent protein kinaseⅡ(CaM KⅡ)-eukaryotic elongation factor 2 (eEF2) and activating the mammalian target of rapamycin (mTOR).Pre-activation of CaMKⅡsignaling within the DG blunted PACAP-induced rapid antidepressant response as well as eEF2-mTOR-brain-derived neurotrophic factor (BDNF) signaling.Finally,acute ketamine treatment upregulated hippocampal PACAP expression,whereas intraDG blockade of PACAP signaling attenuated ketamine’s rapid antidepressant response.Conclusions:Activation of hippocampal PACAP signaling induces a rapid antidepressant response through the regulation of CaMKⅡinhibition-governed eEF2-mTOR-BDNF signaling.

Ethnopharmacological relevancy Zexieyin formula (ZXYF) has been identified to have therapeutic actions of atherosclerosis (AS). It's unknown that whether ZXYF has therapeutic potential of atherosclerosis (AS) with cognitive impairment (CI) and its underlying mechanisms. Aim of the study To elucidate therapeutic effect of ZXYF for AS with CI as well as its underlying mechanisms in AS with CI mice model. Methods and materials To establish AS with CI model, we fed ApoE(-/-) mice with high-fat diet (HFD) for 8 weeks. Oil red O staining (ORO) and Hematoxylin-eosin staining (HE) were used to detect aortic plaque area. Morris water maze (MWM) and Y-maze were used to measure cognitive function and cognitive improvement after administration of ZXYF and atorvastatin (ATO). Network pharmacology was used to screen for potential mechanisms for improving cognitive function. Western blot was used to detect expressions of MAPK, A beta and synaptic proteins in hippocampus. Results HFD caused and accelerated the AS in ApoE(-/-) mice, while it was easier able to produce CI than normal mice. Administration of ZXYF or ATO for 8 weeks significantly reduced aortic plaque area in ORO and HE tests, and improved cognitive abilities in MWM and Y-maze tests. Network pharmacology results showed that MAPK or synaptic proteins were highly associated with CI. HFD contributed to abnormal expressions of MAPK (pERK, pP38, pJNK), NF-kB, synaptic proteins (PSD95, synapsin1) and beta-amyloid (A beta) in hippocampus, which were all reversed by ZXYF. However, ERK and PSD95 expressions were not reversed by ATO in hippocampus. Conclusions ZXYF mitigated AS, further alleviating CI by modulating MAPK signaling, relating to synaptic proteins enhancing and A beta protein decreasing in the hippocampus. This study firstly lit up the new clinical application of ZXYF, which might promote the use of ZXYF in AS and CI patients.

The incidence of Parkinson’s disease (PD) rises rapidly with the increase of age. With the advent of global aging, the number of patients with PD is rising along with the elderly population, especially in China. Previously, we found that Yishen chuchan decoction (YCD), prescribed based on clinical experience, has the potential of alleviating symptoms, delaying the progression, and controlling the development of PD. Nonetheless, the underlying mechanistic role is yet to be explored. Aim. This research examined the possible therapeutic effects of YCD in alleviating PD via a systematic approach with network pharmacology and experimental validation, aiming at providing a new understanding of traditional Chinese medicine management regarding PD. Methods. The chemical structure and properties of YCD were adopted from Traditional Chinese Medicine System Pharmacology Database (TCMSP), SwissADME, PubChem, and PubMed. The potential targets for YCD and PD were identified using Swiss Target Prediction, GeneCard, PubChem, and UniProt. The herbal-component-target network was created via the Cytoscape software. Moreover, by using the STRING database, the protein-protein interaction (PPI) network was screened. Gene function GO and KEGG pathway enrichment analyses were performed via the Metascape database. YCD-medicated Rat Serum from Sprague-Dawley (SD) Rats was prepared, and SH-SY5Y cells were preconditioned with rotenone to develop the PD model. To examine the impact of YCD on these cells and explore the mechanistic role of the p38 mitogen-activated protein kinase (MAPK) pathway, the cells were pretreated with either serum or a p38 MAPK pathway inhibitor. This study employed the Cell Counting Kit (CCK)-8 assay and Hoechst 33342 staining to evaluate the viability and morphological changes induced by the YCD-medicated rat serum on rotenone-treated SH-SY5Y cells. Apoptosis was assessed by Flow cytometry. Immunofluorescence staining assessed the microtubule-associated protein 2 (MAP2) level. Enzyme-linked immunosorbent assay (ELISA) was employed to quantify the concentrations of inflammatory mediators interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). Also, reactive oxygen species (ROS) and superoxide dismutase (SOD) levels were determined. Western Blotting measured the expression of total and phospho-p38 MAPK (p-p38). Results. This study identified 65 active components in YCD, which were found to target 801 specific genes. By screening, 63 potential core targets were identified from a pool of 172 overlapping targets between PD and YCD. These targets were examined by GO and KEGG analyses revealing their substantial correlation to MAPK, PI3K-Akt signaling pathways, positively controlling protein phosphorylation, and pathways of neurodegenerative diseases. SH-SY5Y cells were treated with 2 μM rotenone for 48 h, which reduced cell viability to 50%, and reduced MAP2 expression, increased the rate of apoptosis, oxidative stress, inflammation, and p-p38 expressions. YCD-medicated rat serum significantly improved the viability, reduced the apoptosis rate, and increased the MAP2 expression. YCD-medicated serum increased SOD, reduced ROS and suppressed IL-6, IL-1β and TNF-α levels, thus inhibiting oxidative stress and inflammation in rotenone-treated SH-SY5Y cells. Moreover, YCD-medicated serum substantially lowered the p-p38 expression induced by rotenone. SB203580, a specific inhibitor of p38 MAPK, could also inhibit the p-p38 expression, apoptosis, and restore morphological damage of cells, also improve inflammation and oxidative stress. Conclusion. YCD enhanced cell viability and reduced apoptosis rate, inflammation, and oxidative stress in vitro. These beneficial effects could potentially involve the suppression of p38 pathway and suppressed the phosphorylation of p38 MAPK.

BackgroundAlzheimer's disease (AD) is a serious neurodegenerative disease and brings a serious burden to society and families. Due to lack of effective drugs for the treatment of AD, it's urgent to develop new and effective drug for the treatment of AD.PurposeThe study aimed to investigate the potential of Zexieyin formula (ZXYF), a Chinese medicine formula, for the treatment of AD and its potential mechanism of action.MethodsWe used chronic scopolamine (SCOP) induction mice model and APP/PS1 mice to reveal and confirm ZXYF for the treatment of AD with donepezil (DON) as a positive reference. The learning and memory function were detected by morris water maze test (MWM) and y-maze test. Moreover, western blot and immunofluorescence were used to detect the molecular mechanism of ZXYF for the alleviation of AD in hippocampus. Lastly, pharmacological technology was applied to evaluate AMPA receptor involved in the role of ZXYF in the treatment of AD.ResultsThe results showed that ZXYF could improve memory and learning deficits both in two AD models including scopolamine (SCOP)-induced mice model and APP/PS1mice. Moreover, ZXYF or not DON increased expressions of BrdU/DCX and Ki67 positive cells in dentate gyrus (DG), up-regulated the levels of AMPA subunit type (GluA1) and PKA in hippocampus in SCOP-induced mice model, although ZXYF and DON activated CaMKII, CaMKII-phosphorylation, CREB, CREB-phosphorylation and PSD95 in hippocampus in SCOP-induced mice model. ZXYF also activated CaMKII, CaMKII-phosphorylation and GluA1 in HT22 cells. Furthermore, transient inhibiting AMPA receptor was capable of blocking the effects of ZXYF to treat AD in MWM and suppressing the number of BrdU/DCX positive cells increased by ZXYF in DG in SCOP-induced mice model, but had no effect on the alteration of Ki67 positive cells.ConclusionZXYF had the therapeutic effects on AD-treatment, which activated CaMKII to promote AMPA receptor (GluA1) and subsequently up-regulated PKA/CREB signaling to facilitate neurogenesis to achieve enhanced postsynaptic protein.

Depression is one of the major mental disorders, which seriously endangers human health, brings a serious burden to patients' families. In this study, we intended to further explore the antidepressant-like effect and possible molecular mechanisms of Salidroside (SAL). We built corticosterone (CORT)-induced depressive mice model and used behavioural tests to evaluate depression behaviour. To explore the molecular mechanisms of SAL, we employed a variety of methods such as immunofluorescence, western blot, pharmacological interference, etc. The results demonstrated that SAL both at 25 mg/kg and 50 mg/kg can reduce immobility time in the tail suspension test (TST). At the same time, SAL treatment could restore the reduced sugar water intake preference in the sucrose preference test (SPT) in CORT-induced depressive mice and reduce the immobility time in TST and forced swimming experiments (FST). In addition, SAL treatment reversed the reduction in the number of Ki-67, BrdU, and NeuN in the hippocampus due to CORT treatment. SAL treatment also restored the expression of SIRT1, PGC-1 alpha, brain-derived neurotrophic factor (BDNF) and other proteins in the hippocampus. In addition, after blocking SIRT1 signalling with EX527, we found that the treatment with SAL failed to reduce the immobility time in TST and FST, the level of SIRT1 and PGC-1 alpha activity were correspondingly downregulated, and the expression of DCX and Ki-67 in the hippocampus failed to be activated. These findings suggested that SAL exerts antidepressant-like effects by promoting hippocampal neurogenesis through the SIRT1/PGC-1 alpha signalling pathway.