ZeXieYin formula alleviates atherosclerosis by inhibiting the MAPK/NF-κB signaling pathway in APOE-/- mice to attenuate vascular inflammation and increase plaque stability.

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-1ß, IL-10, IL-6), MAPK/NF-κB pathway proteins, and NLRP3 inflammasomes (NLRP3, Caspase-1) using Western blot. Additionally, the levels of matrix metalloproteinase (MMP)-2 and MMP-9 and α-smooth muscle actin (α-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-κB signaling pathway, and released pro-inflammatory factors such as interleukin (IL)-1ß, IL-1α, 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-κB signaling pathway, which provided a theoretical basis for clinical application and subsequent research.

Zexieyin formula alleviates atherosclerosis with cognitive impairment: A novel role in the treatment of comorbidities and its underlying mechanisms.

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, Aß 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 ß-amyloid (Aß) 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 Aß 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.

ZeXieYin Formula alleviates TMAO-induced cognitive impairment by restoring synaptic plasticity damage.

ETHNOPHARMACOLOGICAL RELEVANCE: Treating cognitive impairment is a challenging and necessary research topic. ZeXieYin Formula (ZXYF), is a traditional herbal formula documented in the book of HuangDiNeiJing. Our previous studies demonstrated the ameliorative effects of ZXYF on atherosclerosis by reducing the plasma trimethylamine oxide (TMAO) level. TMAO is a metabolite of gut microorganisms, our recent research found that the increasing level of TMAO may have adverse effects on cognitive functions. AIM OF THE STUDY: Our study mainly focused on the therapeutic effects of ZXYF on TMAO-induced cognitive impairment in mice and explored its underlying mechanism. MATERIALS AND METHODS: After the TMAO-induced cognitive impairment mice models were established, we applied behavioral tests to estimate the learning and memory ability of the ZXYF intervention mice. Liquid chromatography-mass spectrometry (LC-MS) was used to quantify the TMAO levels in plasma and the brain. The effects of ZXYF on the hippocampal synaptic structure and the neurons were observed by transmission electron microscopy (TEM) and Nissl staining. In addition, western-blotting (WB) and immunohistochemical (IHC) staining were used to detect the level of related proteins in the synaptic structure and further verify the changes in synaptic plasticity and the mTOR pathway after ZXYF administration. RESULTS: Behavioral tests showed that the learning and memory ability of mice impaired after a period of TMAO intervention and ZXYF could alleviate these changes. A series of results showed that ZXYF partly restored the damage of hippocampal synapse and neurons in TMAO-induced mice, at the same time, the expression of synapse-related proteins and mTOR pathway-related proteins were significantly regulated compared with the damage caused by TMAO. CONCLUSION: ZXYF could alleviate TMAO-induced cognitive impairment by improving synaptic function, reducing neuronal damage, regulating synapse-associated proteins, and regulating the mTOR signaling pathway.

PI3K/AKT/SERBP-1 pathway regulates Alisma orientalis beverage treatment of atherosclerosis in APOE-/- high-fat diet mice.

CONTEXT: Previously, we found Alisma orientalis beverage (AOB), a classic traditional Chinese medicine (TCM) formulation, had the potential effect of treating atherosclerosis (AS). The underlying mechanism was still unclear. OBJECTIVE: As an extention of our previous work, to investigate the underlying mechanism of action of AOB in the treatment for AS. MATERIALS AND METHODS: Network pharmacology was conducted using SwissTargetPrediction, GeneCards, DrugBank, Metascape, etc., to construct component-target-pathway networks. In vivo, AS models were induced by a high-fat diet (HFD) for 8 consecutive weeks in APOE-/- mice. After the administration of AOB (3.8 g/kg, i.g.) for 8 weeks, we assessed the aortic plaque, four indicators of blood lipids, and expression of the PI3K/AKT/SREBP-1 pathway in liver. RESULTS: Network pharmacology showed that PI3K/AKT/SREBP-1 played a role in AOB's treatment for AS (PI3K: degree = 18; AKT: degree = 17). Moreover, we found that the arterial plaque area and four indicators of blood lipids were all significantly reversed by AOB treatment in APOE-/- mice fed with HFD (plaque area reduced by about 37.75%). In addition, phosphorylated expression of PI3K/AKT and expression of SREBP-1 were obviously increased in APOE-/- mice fed with HFD, which were all improved by AOB (PI3K: 51.6%; AKT: 23.6%; SREBP-1: 40.0%). CONCLUSIONS: AOB had therapeutic effects for AS by improving blood lipids and inhibition of the PI3K/AKT/SERBP-1 pathway in the liver. This study provides new ideas for the treatment of AS, as well as new evidence for the clinical application of AOB.

Synergistic effects of two naturally occurring iridoids in eliciting a rapid antidepressant action by up-regulating hippocampal PACAP signalling.

BACKGROUND AND PURPOSE: Current mainstream antidepressants have limited efficacy with a delayed onset of action. Yueju, a herbal medicine, has a rapid antidepressant action. Identification of the active ingredients in Yueju and the mechanism/s involved was carried out. EXPERIMENTAL APPROACH: Key molecule/s and compounds involved in this antidepressant action was identified by transcriptomic and HPLC analysis, respectively. Antidepressant effects were evaluated using various behavioural experiments. The signalling involved was assessed using site-directed pharmacological intervention or optogenetic manipulation. KEY RESULTS: Transcriptomic analysis showed that Yueju up-regulated pituitary adenylate cyclase activating polypeptide (PACAP) expression in the hippocampus. Two iridoids, geniposide and shanzhiside methyl ester, were identified and quantified from Yueju. Only co-treatment with both, at an equivalent concentrations found in Yueju, increased PACAP expression and elicited a rapid antidepressant action, which were blocked by intra-dentate gyrus infusion of a PACAP antagonist or optogenetic inactivation of PACAP expressing neurons. Geniposide and shanzhiside methyl ester co-treatment rapidly inhibited CaMKII phosphorylation and enhanced mTOR/4EBP1/P70S6k/BDNF ignalling, while intra-dentate gyrus infusions of a CaMKII activator blunted the rapid antidepressant action and BDNF expression up-regulation induced by the co-treatment. A single co-treatment of them rapidly improved depression-like behaviours and up-regulated hippocampal PACAP signalling in the repeated corticosterone-induced depression model, further confirming the involvement of PACAP. CONCLUSION AND IMPLICATIONS: Geniposide and shanzhiside methyl ester co-treatment had a synergistic rapid onset antidepressant action by triggering hippocampal PACAP activity and associated CaMKII-BDNF signalling. This mechanism could be targeted for development of fast onset antidepressants.

Jianpi Jieyu Decoction, An Empirical Herbal Formula, Exerts Psychotropic Effects in Association With Modulation of Gut Microbial Diversity and GABA Activity.

Background: Recent studies suggest that gut microbiota was associated with the bidirectional gut-brain axis which could modulate neuropsychological functions of the central nervous system. Gut microbiota could produce gamma aminobutyric acid (GABA) that could modulate the gut-brain axis response. Jianpi Jieyu (JPJY) decoction, a traditional Chinese formula, is mainly composed of Astragalus membranaxeus and Radix Pseudostellariae. Although the JPJY decoction has been used to treat the depression in China, the potential action of its antidepressant has not been well understood. Thus this study was aim to investigate the role of JPJY improve gut microbiota homeostasis in the chronic stress induced depressive mice. Methods: The antidepressant effect of JPJY on chronic unpredictable mild stress (CUMS) mice was evaluated by using sucrose preference test, tail suspension test and forced swim test. Fatigue-like behaviors were evaluated using degree of redness, grip strength test, and exhaustive swimming test. The new object recognition test was used to evaluate cognition performance. Fecal samples were collected and taxonomical analysis of intestinal microbial distribution was conducted with 16S rDNA. Serum level of GABA was measured using high performance liquid chromatography (HPLC). The expression of GluR1 and p-Tau protein in the hippocampus was determined using Western blotting. Results: The dose of 9.2 g/kg JPJY produced antidepressant-like effects. JPJY and its major components also modulated gut microbiota diversity in the CUMS mice. Serum level of GABA and the expressions of hippocampal GluR1 and p-Tau were reversed after the administration of JPJY in CUMS mice. Conclusion: JPJY regulates gut microbiota to produce antidepressant-like effect and improve cognition deficit in depressive mice while its molecular mechanism possibly be enhanced NR1 and Tau expression in hippocampus and increased GABA in serum.

Alisma orientalis Beverage Treats Atherosclerosis by Regulating Gut Microbiota in ApoE-/- Mice.

BACKGROUND: Alisma orientalis beverage (AOB) is a Chinese traditional medicine formulated with a diversity of medicinal plants and used for treating metabolic syndrome and atherosclerosis (AS) since time ago. Given the current limited biological research on AOB, the mechanism by which AOB treats AS is unknown. This study investigats the role of AOB-induced gut microbiota regulation in the expansion of AS. METHODS: We established an AS model in male apolipoprotein E-deficient (ApoE-/-) mice that are fed with a high-fat diet (HFD), treated with numerous interventions, and evaluated the inflammatory cytokines and serum biochemical indices. The root of the aorta was stained with oil red O, and the proportion of the lesion area was quantified. Trimethylamine N-oxide (TMAO) and trimethylamine (TMA) levels in serum were evaluated through liquid chromatography with mass spectrometry. Flavin-containing monooxygenase 3 (FMO3) liver protein expression was assessed by Western blotting. 16S rDNA sequencing technique was adopted to establish the changes in the microbiota structure. RESULTS: After 8 weeks of HFD feeding, an inflammatory cytokine, and AS development expression were significantly decreased in mice treated with AOB; the same parameters in the mice treated with the antibiotics cocktail did not change. In the gut microbiota study, mice treated with AOB had a markedly different gut microbiota than the HFD-fed mice. Additionally, AOB also decreased serum TMAO and hepatic FMO3 expression. CONCLUSION: The antiatherosclerotic effects of AOB were found associated with changes in the content of gut microbiota and a reduction in TMAO, a gut microbiota metabolite, suggesting that AOB has potential therapeutic value in the treatment of AS.