Gualou-Xiebai-Banxia-Tang regulates liver-gut axis to ameliorate Metabolic Syndrome in HFD-fed mice.

BACKGROUND: Metabolic syndrome (MetS), characterized by obesity, hyperglycemia, and abnormal blood lipid levels, is the pathological basis of many cardiovascular diseases. Gualou-Xiebai-Banxia-Tang decoction (GT) was first described in the Synopsis of the Golden Chamber, the earliest traditional Chinese medicine (TCM) monograph on diagnosis and treatment of miscellaneous diseases in China. According to TCM precepts, based on its ability to activate yang to release stagnation, activate qi to reduce depression, remove phlegm, and broaden the chest, GT has been used for more than 2,000 years to treat cardiovascular ailments. However, the molecular bases of its therapeutic mechanisms remain unclear. PURPOSE: The aim of this study was to identify lipid- and glucose-related hepatic genes differentially regulated by GT, and to assess GT impact on gut microbiota composition, in mice with high-fat diet (HFD)-induced MetS. STUDY DESIGN AND METHODS: ApoE-/- mice were fed with an HFD for 24 weeks, with or without concurrent GT supplementation, to induce MetS. At the study's end, body weight, visceral fat weight, blood lipid levels, and insulin sensitivity were measured, and histopathological staining was used to evaluate hepatosteatosis and intestinal barrier integrity. Liver transcriptomics was used for analysis of differentially expressed genes in liver and prediction of relevant regulatory pathways. Hepatic lipid/glucose metabolism-related genes and proteins were detected by RT-qPCR and western blotting. Gut microbial composition was determined by 16S rRNA gene sequencing. RESULTS: GT administration reduced MetS-related liver steatosis and weight gain, promoted insulin sensitivity and lipid metabolism, and beneficially modulated gut microbiota composition by decreasing the relative abundance of g_Lachnospiraceae_NK4A136_group and increasing the relative abundance of g_Alistipes. Liver transcriptomics revealed that GT regulated the expression of genes related to lipid and glucose metabolism (Pparγ, Igf1, Gpnmb, and Trem2) and of genes encoding chemokines/chemokine receptors (e.g. Cxcl9 and Cx3cr1). Significant, positive correlations were found for Ccr2, Ccl4, Ccr1, and Cx3cr1 and the g_Lachnospiraceae_NK4A136_group, and between Cxcl9, Ccr2, Ccl4, and Cx3cr1 and g_Desulfovibrio. GT treatment downregulated the protein expressions of SCD1 and CX3CR1 and upregulated the expression of PCK1 protein. CONCLUSION: GT supplementation alleviates HFD-induced MetS in mice by improving hepatic lipid and glucose metabolism. The anti-metabolic syndrome effects of GT may be related to the regulation of the gut-liver axis.

Geniposide ameliorates atherosclerosis by regulating macrophage polarization via perivascular adipocyte-derived CXCL14.

ETHNOPHARMACOLOGICAL RELEVANCE: Gardenia jasminoides Ellis is a traditional Chinese medicine that has been used for treatment of various diseases, including atherosclerosis by clearing heat and detoxication. Geniposide is considered as the effective compounds responsible for the therapeutic efficacy of Gardenia jasminoides Ellis against atherosclerosis. AIM OF THE STUDY: To investigate the effect of geniposide on atherosclerosis burden and plaque macrophage polarization, with focus on its potential impact on CXCL14 expression by perivascular adipose tissue (PVAT). MATERIALS AND METHODS: ApoE-/- mice fed a western diet (WD) were used to model atherosclerosis. In vitro cultures of mouse 3T3-L1 preadipocytes and RAW264.7 macrophages were used for molecular assays. RESULTS: The results revealed that geniposide treatment reduced atherosclerotic lesions in ApoE-/- mice, and this effect was correlated with increased M2 and decreased M1 polarization of plaque macrophages. Of note, geniposide increased the expression of CXCL14 in PVAT, and both the anti-atherosclerotic effect of geniposide, as well as its regulatory influence on macrophage polarization, were abrogated upon in vivo CXCL14 knockdown. In line with these findings, exposure to conditioned medium from geniposide-treated 3T3-L1 adipocytes (or to recombinant CXCL14 protein) enhanced M2 polarization in interleukin-4 (IL-4) treated RAW264.7 macrophages, and this effect was negated after CXCL14 silencing in 3T3-L1 cells. CONCLUSION: In summary, our findings suggest that geniposide protects ApoE-/- mice against WD-induced atherosclerosis by inducing M2 polarization of plaque macrophages via enhanced expression of CXCL14 in PVAT. These data provide novel insights into PVAT paracrine function in atherosclerosis and reaffirm geniposide as a therapeutic drug candidate for atherosclerosis treatment.

Dingxin recipe â ¢ ameliorates hyperlipidemia injury in SD rats by improving the gut barrier, particularly the SCFAs/GPR43 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Dingxin Recipe Ⅲ (DXR Ⅲ) is a traditional Chinese medicine compound used for hyperlipidemia treatment in clinical practice. However, its curative effects and pharmacological mechanisms in hyperlipidemia have not been clarified to date. AIM OF THE STUDY: Studies have demonstrated that gut barrier was strongly implicated in lipid deposition. Based on gut barrier and lipid metabolism, this study examined the effects and molecular mechanisms of DXR Ⅲ in hyperlipidemia. MATERIALS AND METHODS: The bioactive compounds of DXR Ⅲ were detected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, and its effects were evaluated in high-fat diet-fed rats. Specifically, the serum levels of lipids and hepatic enzymes were measured using the appropriate kits; colon and liver sections were obtained for histological analyses; gut microbiota and metabolites were analyzed by 16S rDNA sequencing and liquid chromatography-MS/MS; and the expression of genes and proteins was determined by real-time quantitative polymerase chain reaction and western blotting and immunohistochemistry, respectively. The pharmacological mechanisms of DXR Ⅲ were further explored by fecal microbiota transplantation and short-chain fatty acid (SCFAs)-based interventions. RESULTS: DXR Ⅲ treatment significantly downregulated serum lipid levels, mitigated hepatocyte steatosis and improved lipid metabolism. Moreover, DXR Ⅲ improved the gut barrier, specifically by improving the physical barrier in the colon, causing part composition changes in the gut microbiota, and increasing the serum SCFAs level. DXR Ⅲ also upregulated the expression of colon GPR43/GPR109A. Fecal microbiota transplantation from rats treated with DXR Ⅲ downregulated part hyperlipidemia-related phenotypes, while the SCFAs intervention significantly improved most of the hyperlipidemia-related phenotypes and upregulated the expression of GPR43. Moreover, both DXR Ⅲ and SCFAs upregulated the expression of colon ABCA1. CONCLUSION: DXR Ⅲ protects against hyperlipidemia by improving the gut barrier, particularly the SCFAs/GPR43 pathway.

Generation of p27icreER transgenic mice: A tool for inducible gene expression in supporting cells in the cochlea.

The loss of cochlear hair cells (HCs) is an important cause of sensorineural hearing loss, and finding ways to regenerate HCs would be the ideal way forward for restoring hearing. In this research field, tamoxifen-inducible Cre recombinase (iCreER) transgenic mice and the Cre-loxp system are widely used to manipulate gene expression in supporting cells (SCs), which lie beneath the sensory HCs and are a natural source for HC regeneration. However, many iCreER transgenic lines are of limited utility because they cannot target all subtypes of SCs or they cannot be used in the adult stage. In this study, a new line of iCreER transgenic mice, the p27-P2A-iCreERT2 knock-in mouse strain, was generated by inserting the P2A-iCreERT2 cassette immediately in front of the stop codon of p27, which kept the endogenous expression and function of p27 intact. Using a reporter mouse line with tdTomato fluorescence, we showed that the p27iCreER transgenic line can target all subtypes of cochlear SCs, including Claudius cells. p27-CreER activity in SCs was observed in both the postnatal and the adult stage, suggesting that this mouse strain can be useful for research work in adult cochlear HC regeneration. We then overexpressed Gfi1, Pou4f3, and Atoh1 in p27+ SCs of P6/7 mice using this strain and successfully induced many new Myo7a/tdTomato double-positive cells, further confirming that the p27-P2A-iCreERT2 mouse strain is a new and reliable tool for cochlear HC regeneration and hearing restoration.

Electric Spark Deposition of Antibacterial Silver Coating on Microstructured Titanium Surfaces with a Novel Flexible Brush Electrode.

Infection caused by orthopedic titanium implants, which results in tissue damage, is a key factor in endosseous implant failure. Given the seriousness of implant infections and the limitations of antibiotic therapy, surface microstructures and antimicrobial silver coatings have emerged as prominent research areas and have displayed certain antimicrobial effects. Researchers are now working to combine the two to produce more effective antimicrobial surfaces. However, building robust and homogeneous coatings on complex microstructured surfaces is a tough task due to the limits of surface modification techniques. In this study, a novel flexible electrode brush (silver brush) instead of a traditional hard electrode was designed with electrical discharge machining, which has the ability to adapt to complex groove interiors. The results showed that the use of flexible electrode brush allowed silver to be deposited uniformly in titanium alloy microgrooves. On the surface of Ag-TC4, a uniformly covered deposit was visible, and it slowly released silver ions into a liquid environment. In vitro bacterial assays showed that a Ag-TC4 microstructured surface reduced bacterial adhesion and bacterial biofilm formation, and the antibacterial activity of Ag-TC4 against Staphylococcus aureus and Escherichia coli was 99.68% ± 0.002 and 99.50% ± 0.007, respectively. This research could lay the groundwork for the study of antimicrobial metal bound to microstructured surfaces and pave the way for future implant surface design.

Stigmasterol attenuates hepatic steatosis in rats by strengthening the intestinal barrier and improving bile acid metabolism.

Stigmasterol (ST) has been shown to improve both lipid and bile acid (BA) metabolism. However, the mechanism(s) by which ST prevents dyslipidemia via BA metabolism, and the potential involvement of other regulatory mechanisms, remains unclear. Here, we found that ST treatment effectively alleviates lipid metabolism disorder induced by a high-fat diet (HFD). Moreover, we also show that fecal microbiota transplantation from ST-treated rats displays similar protective effects in rats fed on an HFD. Our data confirm that the gut microbiota plays a key role in attenuating HFD-induced fat deposition and metabolic disorders. In particular, ST reverses HFD-induced gut microbiota dysbiosis in rats by reducing the relative abundance of Erysipelotrichaceae and Allobaculum bacteria in the gut. In addition, ST treatment also modifies the serum and fecal BA metabolome profiles in rats, especially in CYP7A1 mediated BA metabolic pathways. Furthermore, chenodeoxycholic acid combined with ST improves the therapeutic effects in HFD-induced dyslipidemia and hepatic steatosis. In addition, this treatment strategy also alters BA metabolism profiles via the CYP7A1 pathway and gut microbiota. Taken together, ST exerts beneficial effects against HFD-induced hyperlipidemia and obesity with the underlying mechanism being partially related to both the reprogramming of the intestinal microbiota and metabolism of BAs in enterohepatic circulation. This study provides a theoretical basis for further study of the anti-obesity effects of ST and consideration of the gut microbiota as a potential target for the treatment of HFD-induced dyslipidemia.

The Role of Ophiopogonin D in Atherosclerosis: Impact on Lipid Metabolism and Gut Microbiota.

Gut microbiota has been proven to play an important role in many metabolic diseases and cardiovascular disease, particularly atherosclerosis. Ophiopogonin D (OPD), one of the effective compounds in Ophiopogon japonicus, is considered beneficial to metabolic syndrome and cardiovascular diseases. In this study, we have illuminated the effect of OPD in ApoE knockout (ApoE[Formula: see text] mice on the development of atherosclerosis and gut microbiota. To investigate the potential ability of OPD to alleviate atherosclerosis, 24 eight-week-old male ApoE[Formula: see text] mice (C57BL/6 background) were fed a high-fat diet (HFD) for 12 weeks, and 8 male C57BL/6 mice were fed a normal diet, serving as the control group. ApoE[Formula: see text] mice were randomly divided into the model group, OPD group, and simvastatin group ([Formula: see text]= 8). After treatment for 12 consecutive weeks, the results showed that OPD treatment significantly decreased the plaque formation and levels of serum lipid compared with those in the model group. In addition, OPD improved oral glucose tolerance and insulin resistance as well as reducing hepatocyte steatosis. Further analysis revealed that OPD might attenuate atherosclerosis through inhibiting mTOR phosphorylation and the consequent lipid metabolism signaling pathways mediated by SREBP1 and SCD1 in vivo and in vitro. Furthermore, OPD treatment led to significant structural changes in gut microbiota and fecal metabolites in HFD-fed mice and reduced the relative abundance of Erysipelotrichaceae genera associated with cholesterol metabolism. Collectively, these findings illustrate that OPD could significantly protect against atherosclerosis, which might be associated with the moderation of lipid metabolism and alterations in gut microbiota composition and fecal metabolites.

Generation of mature and functional hair cells by co-expression of Gfi1, Pou4f3, and Atoh1 in the postnatal mouse cochlea.

The mammalian cochlea cannot regenerate functional hair cells (HCs) spontaneously. Atoh1 overexpression as well as other strategies are unable to generate functional HCs. Here, we simultaneously upregulated the expression of Gfi1, Pou4f3, and Atoh1 in postnatal cochlear supporting cells (SCs) in vivo, which efficiently converted SCs into HCs. The newly regenerated HCs expressed HC markers Myo7a, Calbindin, Parvalbumin, and Ctbp2 and were innervated by neurites. Importantly, many new HCs expressed the mature and terminal marker Prestin or vesicular glutamate transporter 3 (vGlut3), depending on the subtypes of the source SCs. Finally, our patch-clamp analysis showed that the new HCs in the medial region acquired a large K+ current, fired spikes transiently, and exhibited signature refinement of ribbon synapse functions, in close resemblance to native wild-type inner HCs. We demonstrated that co-upregulating Gfi1, Pou4f3, and Atoh1 enhances the efficiency of HC generation and promotes the functional maturation of new HCs.

Ferulic Acid Ameliorates Atherosclerotic Injury by Modulating Gut Microbiota and Lipid Metabolism.

Atherosclerosis is a leading cause of death worldwide. Recent studies have emphasized the significance of gut microbiota and lipid metabolism in the development of atherosclerosis. Herein, the effects and molecular mechanisms involving ferulic acid (FA) was examined in atherosclerosis using the ApoE-knockout (ApoE-∕-, c57BL/6 background) mouse model. Eighteen male ApoE-/- mice were fed a high-fat diet (HFD) for 12 weeks and then randomly divided into three groups: the model group, the FA (40 mg/kg/day) group and simvastatin (5 mg/kg/day) group. As results, FA could significantly alleviate atherosclerosis and regulate lipid levels in mice. Liver injury and hepatocyte steatosis induced by HFD were also mitigated by FA. FA improved lipid metabolism involving up-regulation of AMPKα phosphorylation and down-regulation of SREBP1 and ACC1 expression. Furthermore, FA induced marked structural changes in the gut microbiota and fecal metabolites and specifically reduced the relative abundance of Fimicutes, Erysipelotrichaceae and Ileibacterium, which were positively correlated with serum lipid levels in atherosclerosis mice. In conclusion, we demonstrate that FA could significantly ameliorate atherosclerotic injury, which may be partly by modulating gut microbiota and lipid metabolism via the AMPKα/SREBP1/ACC1 pathway.

Dingxin Recipe IV attenuates atherosclerosis by regulating lipid metabolism through LXR-α/SREBP1 pathway and modulating the gut microbiota in ApoE-/- mice fed with HFD.

ETHNOPHARMACOLOGICAL RELEVANCE: Dingxin Recipe (DXR) is a traditional Chinese medicine formula that has been reported to be effective and safe treatment for cardiovascular diseases, such as arrhythmias, coronary heart disease. Dingxin Recipe IV (DXR IV) was further improved from the DXR according to the traditional use. However, the mechanism of DXR IV in atherosclerosis is unclear. AIM OF THE STUDY: This study aimed to illustrate whether DXR IV improve atherosclerosis through modulating the lipid metabolism and gut microbiota in atherosclerosis mice. MATERIALS AND METHODS: 40 male ApoE-/- mice were fed on HFD for 12 weeks and were then treated with DXR IV (1.8, 0.9, or 0.45 g/kg/d) for another 12 weeks. The decroation of DXR IV contains four traditional Chinese medicines: the dried rhizome of Coptis chinensis Franch. (15.09%), the root of Salvia miltiorrhiza Bunge (28.30%), the seed of Ziziphus jujuba Mill. (37.74%) and the fruiting body of Ganoderma lucidum (Leyss.ex Fr.) Karst. (18.87%). 8 male c57BL/6 mice fed a normal diet served as control group. The atherosclerotic plaque was quantified by oil-red O staining and masson trichrome staining. Mice feces were collected. The gut micobiota were detected by 16S rRNA gene sequencing and fecal metabolites were analyzed by 1H NMR spectroscopy. The effect of DXR IV on blood lipids (TG, TC, LDL-C, HDL-C) was investigated. The lipid metabolism related genes were determined by RT-qPCR and western blotting respectively. RESULTS: DXR IV exerted the anti-atherosclerosis effect by inhibiting the excessive cholesterol deposition in aorta and regulating the level of TG, TC, LDL-C and HDL-C. The composition of gut microbiota was changed. Interestingly, the relative abundance of Muribaculaceae and Ruminococcaceae increased after DXR IV administration, whereas the abundance of Erysipelotrichaceae decreased, which have been beneficial to lipid metabolism. Nine potential metabolic biomarkers, including acetate, butyrate, propionate, alanine, succinate, valerate, xylose, choline, glutamate, were identified, which were related to fatty acid metabolism. Further, the pathway of fatty acid was detected by the RT-qPCR and western blotting. Compared with model group, the level of LXR-α and SREBP1 decreased significantly in DXR IV group while LXR-ß, SREBP2 showed no statistical significance. It indicated that DXR IV modulated lipid metabolism by LXR-α/SREBP1 but not LXRß and SREBP2. CONCLUSIONS: DXR IV exhibits potential anti-atherosclerosis effect, which is closely related to lipid metabolism and the gut microbiota. This study may provide novel insights into the mechanism of DXR IV on atherosclerosis and a basis for promising clinical usage.

Mimic Cochlear Implant Surgery-Induced Cochlear Infection Fails to Further Damage Auditory Pathway in Deafened Guinea Pigs.

BACKGROUND Kanamycin and subsequent furosemide administration was applied to the healthy guinea pigs to induce deafness. MATERIAL AND METHODS Of the deafened guinea pigs, 10 were further infused with anti-infection procedures (Group B) and the other 10 animals did not undergo anti-infection procedures (Group C). In Group B, the deafened animals were able to restore cochlear and middle ear functions following the anti-infection procedure. In Group C, all animals developed cochlear and middle ear infections. RESULTS Compared to the healthy guinea pigs, hair cells and spiral ganglion neurons (SGN) of deafened animals (in Group B and Group C) were severely damaged. SGN density of deafened animals was significantly lower than that of healthy control animals in all ear turns except the basal turn. There was no significant difference between Group B and Group C in SGN density. The average optical density value of neurofilaments of deafened animals was also significantly decreased after the ototoxic drug administration. Notably, the density of the neurons in the cochlear nucleus region (CNR) of the brainstem were not significantly different between the healthy control guinea pigs and deafened animals. CONCLUSIONS Mimic cochlear implant surgery-induced cochlear infection caused no significant damage to the auditory pathway in ototoxic drug-induced deafened guinea pigs.

Celastrol enhances Atoh1 expression in inner ear stem cells and promotes their differentiation into functional auditory neuronal-like cells.

We aimed to investigate the beneficial effect of Celastrol on inner ear stem cells and potential therapeutic value for hearing loss. The inner ear stem cells were isolated and characterized from utricular sensory epithelium of adult mice. The stemness was evaluated by sphere formation assay. The relative expressions of Atoh1, MAP-2 and Myosin VI were measured by RT-PCR and immunoblotting. The up-regulation of MAP-2 was also analysed with immunofluorescence. The in vitro neuronal excitability was interrogated by calcium oscillation. The electrophysiological property was determined by inward current recorded on patch clamp. Our results demonstrated that Celastrol treatment significantly improved the viability and proliferation of mouse inner ear stem cells, and facilitated sphere formation. Moreover, Celastrol stimulated differentiation of mouse inner ear stem cells to neuronal-like cells and enhanced neural excitability. Celastrol also enhanced neuronal-like cell identity in the inner ear stem cell derived neurons, as well as their electrophysiological function. Most notably, these effects were apparently associated with the upregulation of Atoh1 in response to Celastrol treatment. Celastrol showed beneficial effect on inner ear stem cells and held therapeutic promise against hearing loss.

Junctional E-cadherin/p120-catenin Is Correlated with the Absence of Supporting Cells to Hair Cells Conversion in Postnatal Mice Cochleae.

Notch inhibition is known to generate supernumerary hair cells (HCs) at the expense of supporting cells (SCs) in the mammalian inner ear. However, inhibition of Notch activity becomes progressively less effective at inducing SC-to-HC conversion in the postnatal cochlea and balance organs as the animal ages. It has been suggested that the SC-to-HC conversion capacity is inversely correlated with E-cadherin accumulation in postnatal mammalian utricles. However, whether E-cadherin localization is linked to the SC-to-HC conversion capacity in the mammalian inner ear is poorly understood. In the present study, we treated cochleae from postnatal day 0 (P0) with the Notch signaling inhibitor DAPT and observed apparent SC-to-HC conversion along with E-cadherin/p120ctn disruption in the sensory region. In addition, the SC-to-HC conversion capacity and E-cadherin/p120ctn disorganization were robust in the apex but decreased toward the base. We further demonstrated that the ability to regenerate HCs and the disruption of E-cadherin/p120ctn concomitantly decreased with age and ceased at P7, even after extended DAPT treatments. This timing is consistent with E-cadherin/p120ctn accumulation in the postnatal cochleae. These results suggest that the decreasing capacity of SCs to transdifferentiate into HCs correlates with E-cadherin/p120ctn localization in the postnatal cochleae, which might account for the absence of SC-to-HC conversion in the mammalian cochlea.

Therapeutic value of nerve growth factor in promoting neural stem cell survival and differentiation and protecting against neuronal hearing loss.

Nerve growth factor (NGF) is a neurotrophic factor that modulates survival and differentiation of neural stem cells (NSCs). We investigated the function of NGF in promoting growth and neuronal differentiation of NSCs isolated from mouse cochlear tissue, as well as its protective properties against gentamicin (GMC) ototoxicity. NSCs were isolated from the cochlea of mice and cultured in vitro. Effect of NGF on survival, neurosphere formation, and differentiation of the NSCs, as well as neurite outgrowth and neural excitability in the subsequent in vitro neuronal network, was examined. Mechanotransduction capacity of intact cochlea and auditory brainstem response (ABR) threshold in mice were also measured following GMC treatment to evaluate protection using NGF against GMC-induced neuronal hearing loss. NGF improved survival, neurosphere formation, and neuronal differentiation of mouse cochlear NSCs in vitro, as well as promoted neurite outgrowth and neural excitability in the NSC-differentiated neuronal culture. In addition, NGF protected mechanotransduction capacity and restored ABR threshold in gentamicin ototoxicity mouse model. Our study supports a potential therapeutic value of NGF in promoting proliferation and differentiation of NSCs into functional neurons in vitro, supporting its protective role in the treatment of neuronal hearing loss.