Fufang Zhenzhu Tiaozhi (FTZ) suppression of macrophage pyroptosis: Key to stabilizing rupture-prone plaques.

BACKGROUND: Research on the Chinese herbal formula Fufang Zhenzhu Tiaozhi (FTZ) has demonstrated its effectiveness in treating hyperlipidemia and glycolipid metabolic disorders. Additionally, FTZ has shown inhibitory effects on oxidative stress, regulation of lipid metabolism, and reduction of inflammation in these conditions. However, the precise mechanisms through which FTZ modulates macrophage function in atherosclerosis remain incompletely understood. Therefore, this study aims to investigate whether FTZ can effectively stabilize rupture-prone plaques by suppressing macrophage pyroptosis and impeding the development of M1 macrophage polarization in ApoE-/- mice. METHODS: To assess the impact of FTZ on macrophage function and atherosclerosis in ApoE-/- mice, we orally administered FTZ at a dosage of 1.2 g/kg body weight daily for 14 weeks. Levels of interleukin-18 and interleukin-1ß were quantified using ELISA kits to gauge FTZ's influence on inflammation. Total cholesterol content was measured with a Cholesterol Assay Kit to evaluate FTZ's effect on lipid metabolism. Aortic tissues were stained with Oil Red O, and immunohistochemistry techniques were applied to assess atherosclerotic lesions and plaque stability. To evaluate the effects of FTZ on macrophage pyroptosis and oxidative damage, immunofluorescence staining was utilized. Additionally, we conducted an analysis of protein and mRNA expression levels of NLRP3 inflammasome-related genes and macrophage polarization-related genes using RT-PCR and western blotting techniques. RESULTS: This study illustrates the potential therapeutic effectiveness of FTZ in mitigating the severity of atherosclerosis and improving serum lipid profiles by inhibiting inflammation. The observed enhancements in atherosclerosis severity and inflammation can be attributed to the suppression of NLRP3 inflammasome activity and M1 polarization by FTZ. CONCLUSION: The current findings indicate that FTZ provides protection against atherosclerosis, positioning it as a promising candidate for novel therapies targeting atherosclerosis and related cardiovascular diseases.

Tianhuang formula ameliorates liver fibrosis by inhibiting CCL2-CCR2 axis and MAPK/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: In the progression of chronic liver diseases, liver fibrosis is a reversible pathophysiologic event for liver diseases prognosis and risk of cirrhosis. Liver injury factors of different etiologies mediate this process. There is still a lack of effective medications for treating liver fibrosis. Additionally, the ameliorative effects of traditional herbs on liver fibrosis have been commonly reported. Tianhuang formula (THF) is a drug combination consisting of 2 traditional Chinese herbs, which has been showing significant improvement in metabolic liver diseases. However, the hepatoprotective effect and mechanism of THF in ameliorating liver fibrosis are still unclear. AIM OF THE STUDY: This study aimed to investigate the effects of THF on carbon tetrachloride (CCl4)-induced and methionine-choline-deficient (MCD) diet-induced liver fibrosis model and to reveal the potential mechanisms. It can provide experimental evidence for THF as a therapeutic candidate for liver fibrosis. MATERIALS AND METHODS: In this study, CCl4-induced mice were treated with THF (80 mg/kg, 160 mg/kg) or Fuzheng Huayu (FZHY) capsules (4.8 g/kg) for 6 weeks. MCD-induced mice received the same doses of THF or FZHY for 4 weeks. FZHY is used as a comparative study in these two models. Following that, using kit reagents detected changes in relevant serum and liver biochemical indicators. Histological changes in mouse liver were measured by staining of H&E and Sirius Red. The markers expression of liver fibrosis and inflammation were detected using qRT-PCR, western blotting and immunohistochemical staining analysis. The potential regulatory mechanism of THF to ameliorate liver fibrosis was performed by RNA-sequencing analysis. Finally, the analysis results were verified by immunofluorescence co-staining, qRT-PCR and western blotting. RESULTS: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hepatic triglyceride (TG) levels in CCl4 and MCD-induced liver fibrosis mice were significantly improved after THF treatment. Meanwhile, the expression of fibrosis and inflammation markers were significantly suppressed. Furthermore, THF downregulated the expression of the macrophage marker CD68. According to RNA-sequencing analysis, we found the CCL2-CCR2 axis and MAPK/NF-κB as the potential signaling pathway for THF against liver fibrosis. CONCLUSION: This study revealed that THF ameliorated liver injury, inflammation and fibrotic process by inhibiting CCL2-CCR2 axis and its downstream MAPK/NF-κB signaling pathway.

Fufang Zhenzhu Tiaozhi (FTZ) capsule ameliorates diabetes-accelerated atherosclerosis via suppressing YTHDF2-mediated m6A modification of SIRT3 mRNA.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang Zhenzhu TiaoZhi (FTZ), a Chinese medicinal decoction, has continuously been used to treat metabolic syndrome. Atherosclerosis is the main pathological basis of cardiovascular disease. The N6 methyladenosine (m6A) modification is a highly dynamic and reversible process involving a variety of important biological processes. AIM OF THE STUDY: Here, we investigated the therapeutic effects and mechanism of FTZ in diabetes-accelerated atherosclerosis. MATERIALS AND METHODS: Doppler ultrasonography was used to examine the carotid intima-media thickness and plaque area in diabetic atherosclerosis patients. HFD mice were injected with streptozotocin to induce diabetes. HE and Oil red O staining were used to assess the effect of FTZ on lipid deposition. HUVECs were induced with HG/ox-LDL as a model of diabetic atherosclerosis. Furthermore, application of m6A methylation level kit, qRT-PCR, Western blot, tunel staining, reactive oxygen species staining and mPTP staining were performed to analyze the detailed mechanism. RESULTS: Clinical trials of FTZ have shown obvious effect of lowering blood glucose and blood lipids. These effects were reversed after FTZ intervention. Compared with the control, lipid deposition decreased significantly after FTZ administration. FTZ reduced endothelial cell apoptosis. At the same time, we found that FTZ reversed the increase of methylation reader YTHDF2 caused by ox-LDL treatment. Subsequently, we discovered that YTHDF2 degraded SIRT3 mRNA, leading to endothelial cell apoptosis and oxidative stress. CONCLUSION: FTZ attenuated diabetes-accelerated atherosclerosis by decreasing blood glucose and serum lipids levels, and increased endothelial cell antioxidant capacity, inhibited endothelial cell apoptosis via inhibiting YTHDF2-mediated m6A modification of SIRT3 mRNA, which reduced mRNA degradation.

Metaflammation in glucolipid metabolic disorders: Pathogenesis and treatment.

The public health issue of glucolipid metabolic disorders (GLMD) has grown significantly, posing a grave threat to human wellness. Its prevalence is rising yearly and tends to affect younger people. Metaflammation is an important mechanism regulating body metabolism. Through a complicated multi-organ crosstalk network involving numerous signaling pathways such as NLRP3/caspase-1/IL-1, NF-B, p38 MAPK, IL-6/STAT3, and PI3K/AKT, it influences systemic metabolic regulation. Numerous inflammatory mediators are essential for preserving metabolic balance, but more research is needed to determine how they contribute to the co-morbidities of numerous metabolic diseases. Whether controlling the inflammatory response can influence the progression of GLMD determines the therapeutic strategy for such diseases. This review thoroughly examines the role of metaflammation in GLMD and combs the research progress of related therapeutic approaches, including inflammatory factor-targeting drugs, traditional Chinese medicine (TCM), and exercise therapy. Multiple metabolic diseases, including diabetes, non-alcoholic fatty liver disease (NAFLD), cardiovascular disease, and others, respond therapeutically to anti-inflammatory therapy on the whole. Moreover, we emphasize the value and open question of anti-inflammatory-based means for treating GLMD.

Purification Process and In Vitro and In Vivo Bioactivity Evaluation of Pectolinarin and Linarin from Cirsium japonicum.

Pectolinarin and linarin are two major flavone O-glycosides of Cirsium japonicum, which has been used for thousands of years in traditional Chinese medicine. Pharmacological research on pectolinarin and linarin is meaningful and necessary. Here, a process for the purification of pectolinarin and linarin from C. japonicum was established using macroporous resin enrichment followed by prep-HPLC separation. The results show the purity of pectolinarin and linarin reached 97.39% and 96.65%, respectively. The in vitro bioactivities result shows the ORAC values of pectolinarin and linarin are 4543 and 1441 µmol TE/g, respectively, meanwhile their inhibition rate of BSA-MGO-derived AGEs is 63.58% and 19.31% at 2 mg/mL, which is 56.03% and 30.73% in the BSA-fructose system, respectively. The COX-2 inhibition rate at 50 µg/mL of linarin and pectolinarin reached 55.35% and 40.40%, respectively. Furthermore, the in vivo bioassay combining of histopathologic evaluation and biochemical analysis of liver glutamic oxaloacetic transaminase, serum creatinine and TNF-α show pectolinarin can alleviate lipopolysaccharide (LPS)-induced acute liver and kidney injury in mice. Metabolomics analysis shows that pectolinarin attenuates LPS-challenged liver and kidney stress through regulating the arachidonic acid metabolism and glutathione synthesis pathways. Collectively, our work presents a solid process for pectolinarin and linarin purification and has discovered a promising natural therapeutic agent-pectolinarin.

Tianhuang formula reduces the oxidative stress response of NAFLD by regulating the gut microbiome in mice.

Background: The gut microbiome affects the occurrence and development of NAFLD, but its mechanism has not yet been fully elucidated. Chinese medicine is a new treatment strategy to improve NAFLD by regulating the gut microbiome. Tianhuang formula (TH) has been proved to have a lipid-lowering effect in which constituents of ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rb, ginsenoside Re, and ginsenoside R1 from Panax notoginseng and berberine, palmatine, and coptisine from Coptis chinensis have low drug permeability, which results in poor intestinal absorption into the human body, and are thus able to come into contact with the gut microflora for a longer time. Therefore, it might be able to influence the gut microbial ecosystem, but it still needs to be investigated. Method: The characteristics of the gut microbiome were represented by 16S rRNA sequencing, and the metabolites in intestinal contents and liver were discovered by non-targeted metabolomics. Correlation analysis and fermentation experiments revealed the relationship between the gut microbiome and metabolites. Blood biochemical indicators, liver function indicators, and oxidation-related indicators were assayed. H&E staining and Oil Red O staining were used to analyze the characteristics of hepatic steatosis. RT-qPCR and western blotting were used to detect the expression of genes and proteins in liver tissues, and fecal microbial transplantation (FMT) was performed to verify the role of the gut microbiome. Results: Gut microbiome especially Lactobacillus reduced, metabolites such as 5-Methoxyindoleacetate (5-MIAA) significantly reduced in the liver and intestinal contents, the level of hepatic GSH and SOD reduced, MDA increased, and the protein expression of Nrf2 also reduced in NAFLD mice induced by high-fat diet (HFD). The normal diet mice transplanted with NAFLD mice feces showed oxidative liver injury, indicating that the NAFLD was closely related to the gut microbiome. TH and TH-treated mice feces both can reshape the gut microbiome, increase the abundance of Lactobacillus and the content of 5-MIAA in intestinal contents and liver, and improve oxidative liver injury. This indicated that the effect of TH improving NAFLD was related to the gut microbiome, especially Lactobacillus. 5-MIAA, produced by Lactobacillus, was proved with fermentation experiments in vitro. Further experiments proved that 5-MIAA activated the Nrf2 pathway to improve oxidative stress in NAFLD mice induced by HFD. TH reshaped the gut microbiome, increased the abundance of Lactobacillus and its metabolite 5-MIAA to alleviate oxidative stress, and improved NAFLD. Conclusion: The study has demonstrated a mechanism by which the gut microbiome modulated oxidative stress in NAFLD mice induced by HFD. The traditional Chinese medicine TH improved NAFLD by regulating the gut microbiome, and its mechanism was related to the "Lactobacillus-5-MIAA-Nrf2" pathway. It provided a promising way for the intervention of NAFLD.

Gut microbiota combined with metabolomics reveals the metabolic profile of the normal aging process and the anti-aging effect of FuFang Zhenshu TiaoZhi(FTZ) in mice.

The aging process is accompanied by changes in the gut microbiota and metabolites. This study aimed to reveal the relationship between gut microbiota and the metabolome at different ages, as well as the anti-aging effect of FTZ, which is an effective clinical prescription for the treatment of hyperlipidemia and diabetes. METHODS: In the present study, mice were randomly divided into different age and FTZ treatment groups. The aging-relevant behavioral phenotype the levels of blood glucose, cholesterol, triglycerides, low density lipoprotein cholesterol, free fatty acids, high density lipoprotein-cholesterol and cytokine TNF-α,IL-6, IL-8 in the serum were measured. Changes of serum metabolties were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-Q-TOF/MS). Gut microbiota were identified using 16S rDNA sequencing. RESULTS: Our results indicated that with age, the aging-relevant behavioral phenotype appeared, glucose and lipid metabolism disordered, secretion levels of cytokine TNF-α, IL-6 and IL-8 increased.The Firmicutes/Bacteroidetes ratio changed with age, first increasing and then decreasing, and the microbial diversity and the community richness of the aging mice were improved by FTZ. The abundance of opportunistic bacteria decreased (Lactobacillus murinus, Erysipelatoclostridium), while the levels of protective bacteria such as Butyricimonas, Clostridium and Akkermansia increased. Metabolic analysis identified 24 potential biomarkers and 10 key pathways involving arachidonic acid metabolism, phospholipid metabolism, fatty acid metabolism, taurine and hypotaurine metabolism. Correlation analysis between the gut microbiota and biomarkers suggested that the relative abundance of protective bacteria was negatively correlated with the levels of leukotriene E4, 20-HETE and arachidonic acid, which was different from protective bacteria. CONCLUSION: Shifts of gut microbiota and metabolomic profiles were observed in the mice during the normal aging process, and treatment with FTZ moderately corrected the aging, which may be mediated via interference with arachidonic acid metabolism, sphingolipid metabolism, glycerophospholipid metabolism, taurine and hypotaurine metabolism and gut microbiota in mice.

Untargeted Metabolomics Reveals the Protective Effect of Fufang Zhenshu Tiaozhi (FTZ) on Aging-Induced Osteoporosis in Mice.

Fufang Zhenzhu Tiaozhi (FTZ), as an effective traditional Chinese medicine, has been prescribed for more than 20 years. It has proven clinical efficacy as a prescription for patients with dyslipidemia, glucocorticoid- and high-fat-induced osteoporosis, but its effect on osteoporosis induced by aging is still unclear. The aim of this study was to investigate the anti-osteoporosis effect of FTZ in aging mice and revealed its biochemical action mechanism using metabolomics. Model of primary osteoporosis induced by aging was established. The mice in treatment group received a therapeutic dose of oral FTZ extract once daily during the experiment. The model and control groups received the corresponding volume of oral normal saline solution. Plasma samples of all three groups were collected after 12 weeks. Clinical biochemical parameters and biomechanics were determined in the osteoporosis model induced by normal aging to evaluate anti-osteoporosis effect of FTZ. Ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was used to analyze metabolic changes. The changes of histomorphometric and biomechanic parameters of femurs, as well as osteoblast and osteoclast activity indicated that FTZ administration reduced the risk of osteoporosis. Partial least squares discriminant analysis (PLS-DA) score plot revealed a clear separation trend between model and controls. Moreover, PLS-DA score plot indicated the anti-osteoporosis effect of FTZ with sphingosine 1-phosphate, LPA (16:0) and arachidonic acid (AA) among key biomarkers. The pivotal pathways revealed by pathway analysis including sphingolipid metabolism, glycerophospholipid metabolism, and AA metabolism. The mechanism by which FTZ reduces the risk of primary age-related osteoporosis in mice might be related to disorders of the above-mentioned pathways. FTZ has a protective effect against osteoporosis induced by aging, which may be mediated via interference with sphingolipid, glycerophospholipid, and AA metabolisms in mice.

Potential synergistic effects of Chinese herbal prescription FTZ components detected in blood towards hepatic lipid-modulating targets.

OBJECTIVE: Our goal in this study aims to explain the polypharmacological mechanism at the molecular level responsible for the effectiveness of a traditional Chinese medicine (TCM) prescription FTZ to treat hyperlipidemia and related disease. DESIGN: By MDL(®) ISIS_Base 2.5, we constructed a compound database based on the FTZ constituents, which were detected in the rat serum after oral administration of the TCM through ultra-performance liquid chromatography/quadruple-time-of-flight mass-spectrometry (UPLC/Q-TOF-MS/MS) method. After validation of the virtual docking system, we used molecular screening by LigandFit which is a computational method for the shape-directed rapid docking of ligands to target protein active sites, to investigate the interactions between the components in database and lipid-modulating targets in the liver. RESULTS: In the prescription FTZ ingredients, there were sixteen constituents including jatrorrhizine, etc. showed potential effects towards the hyperlipidemia-related targets: HMG-CoA reductase (HMGR), squalene synthase (SQS), oxidosqualene cyclase (OSC), cholesteryl ester transfer protein (CETP), liver X receptor (LXR), farnesoid X receptor (FXR) and peroxisome proliferator-activated receptors (PPARα and PPARγ). Among the eight herbs in prescription FTZ, Rhizoma Coptidis (RC) plays the most important role in whole effect from FTZ on hyperlipidemia related disease. CONCLUSIONS: Our research demonstrated that Chinese medicine formula FTZ has multi-target synergistic effect on hyperlipidemia and suggests the pharmacodynamic material basis could be jatrorrhizine, berberrubine, berberine and salidroside.

Hypocholesterolemia of Rhizoma Coptidis alkaloids is related to the bile acid by up-regulated CYP7A1 in hyperlipidemic rats.

This study is to investigate the cholesterol-lowering effect and the new mode of action of coptis alkaloids on high lipid diet-induced hyperlipidemic rats. Coptis alkaloids extract (CAE) was prepared by alcohol extraction from Rhizoma Coptidis that have been quality-controlled according to the protocol. The cholesterol-lowering effect of CAE was evaluated on SD rats fed with high-lipid diet. Serum level of lipid, Bile acid and cholesterol in the liver and feces of the rats were measured using colorimetric assay kit. RT-PCR and Western blot were used to analyze the mRNA and protein expression of cholesterol metabolism-related genes including cholesterol 7α-hydroxylase (CYP7A1), peroxisome proliferator-activated receptor-alpha (PPARα) and farnesoid X receptor (FXR) in the livers of the rats. A HPLC analysis was used to assess the activity of CYP7A1. The results showed that CAE reduced the levels of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C). CYP7A1 gene expression and its activity was up-regulated dose-dependently accompanying with the increased level of bile acid and the reduced cholesterol level in the livers of the CAE treated hyperlipidemic rats. Meanwhile, the mRNA expression of PPARα was also up-regulated in dose-dependent way accompanying the down-modulation of the FXR mRNA expression in the livers of the CAE treated hyperlipidemic rats. The results indicate that the cholesterol-lowering effect of coptis alkaloid extract is at least partly attributed to its promoting the cholesterol conversion into bile acids by up-regulating the gene expression of CYP7A1 and thus increasing its activity in the liver of the hyperlipidemic rats, which might related to the positive regulation of PPARα and the negative modulation of FXR.

Effects of Fufang Zhenzhu Tiaozhi Prescription (), A Chinese herbal preparation, on atherosclerosis in ApoE-/- mice and related mechanisms.

OBJECTIVE: The present study was designed to observe the effects and the possible mechanisms of Fufang Zhenzhu Tiaozhi Prescription (, FZT), a Chinese herbal preparation, on atherosclerosis in apolipoprotein E deficient (ApoE-/-) mice. METHODS: ApoE-/- mice were randomized to groups orally administrated without or with FZT (4.5 and 9 g crude drug/kg body weight, respectively). Atherosclerotic plaques, lipids profifiles in serum, aortic cholesterol content, serum indices of oxidative stress were measured, and mRNA expressions of scavenger receptors CD36 and scavenger receptor-A (SR-A) in aorta were analyzed. RESULTS: FZT dose-dependently suppressed the atherosclerotic plaques and reduced the cholesterol contents in aorta (P<0.05, P<0.01). In addition, FZT decreased the levels of total cholesterol and triglyceride in serum (P<0.05, P<0.01), inhibited the production of oxidized low density lipoprotein and malonaldehyde, and increased the superoxide dismutase activity in serum (P<0.05 or P<0.01). Furthermore, FZT down-regulated the mRNA expressions of CD36 and SR-A in the aorta (P<0.05, P<0.01). CONCLUSIONS: FZT reduced the atherosclerotic formation in ApoE-/- mice. The mechanisms might be related to the decrease of serum lipid level, the improvement of oxidative stress and the down-regulated expressions of scavenger receptors CD36 and SR-A, consequently reducing the foam cell formation, the hallmark of early atherosclerosis.