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.

The Chinese herbal medicine Fufang Zhenzhu Tiaozhi ameliorates diabetic cardiomyopathy by regulating cardiac abnormal lipid metabolism and mitochondrial dynamics in diabetic mice.

Diabetic cardiomyopathy (DCM) is an important complication leading to the death of patients with diabetes, but there is no effective strategy for clinical treatments. Fufang Zhenzhu Tiaozhi (FTZ) is a patent medicine that is a traditional Chinese medicine compound preparation with comprehensive effects for the prevention and treatment of glycolipid metabolic diseases under the guidance of "modulating liver, starting pivot and cleaning turbidity". FTZ was proposed by Professor Guo Jiao and is used for the clinical treatment of hyperlipidemia. This study was designed to explore the regulatory mechanisms of FTZ on heart lipid metabolism dysfunction and mitochondrial dynamics disorder in mice with DCM, and it provides a theoretical basis for the myocardial protective effect of FTZ in diabetes. In this study, we demonstrated that FTZ protected heart function in DCM mice and downregulated the overexpression of free fatty acids (FFAs) uptake-related proteins cluster of differentiation 36 (CD36), fatty acid binding protein 3 (FABP3) and carnitine palmitoyl transferase 1 (CPT1). Moreover, FTZ treatment showed a regulatory effect on mitochondrial dynamics by inhibiting mitochondrial fission and promoting mitochondrial fusion. We also identified in vitro that FTZ could restore lipid metabolism-related proteins, mitochondrial dynamics-related proteins and mitochondrial energy metabolism in PA-treated cardiomyocytes. Our study indicated that FTZ improves the cardiac function of diabetic mice by attenuating the increase in fasting blood glucose levels, inhibiting the decrease in body weight, alleviating disordered lipid metabolism, and restoring mitochondrial dynamics and myocardial apoptosis in diabetic mouse hearts.

FTZ promotes islet ß-cell regeneration in T1DM mice via the regulation of nuclear proliferation factors.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang-Zhenzhu-Tiaozhi capsule (FTZ), a Traditional Chinese Medicine (TCM) patent prescription commonly used in clinical practice, has a significant curative effect on hyperglycemia and hyperlipidemia. Previous studies have shown that FTZ can treat diabetes, but the effect of FTZ on ß-cell regeneration needs to be further explored in T1DM mice. AIM OF THE STUDY: The aim is to investigate the role of FTZ in promoting ß-cell regeneration in T1DM mice, and to further explore its mechanism. MATERIALS AND METHODS: C57BL/6 mice were used as control. NOD/LtJ mice were divided into the Model group and FTZ group. Oral glucose tolerance, fasting blood glucose, and fasting insulin level were measured. Immunofluorescence staining was used to detect the level of ß-cell regeneration and the composition of α-cells and ß-cells in islets. Hematoxylin and eosin staining was used to detect the infiltration degree of inflammatory cells. The apoptosis of islet cells was detected by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling. Western blotting was used to detect the expression levels of Pancreas/duodenum homeobox protein 1 (PDX-1), V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA), and Neurogenin-3 (NGN3). RESULTS: FTZ could increase insulin levels and reduce the glucose level of T1DM mice and promote ß-cell regeneration. FTZ also inhibited the invasion of inflammatory cells and the islet cell apoptosis, and maintained the normal composition of islet cells, thus preserving the quantity and quality of ß-cells. Furthermore, FTZ promoting ß-cell regeneration was accompanied by increasing the expression of PDX-1, MAFA, and NGN3. CONCLUSION: FTZ can restore the insulin-secreting function of the impaired pancreatic islet, improve blood glucose level, possibly via the enhancing ß cell regeneration via upregulation of PDX-1, MAFA, and NGN3 in T1DM mice, and may be a potential therapeutic drug for T1DM.

Fufang-zhenzhu-tiaozhi formula protects islet against injury and promotes ß cell regeneration in diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang-zhenzhu-tiaozhi formula (FTZ) is a patented preparation of traditional Chinese medicine that has been used to treat hyperglycemia and hyperlipidemia in the clinic for almost 10 years. Our previous study had demonstrated that FTZ can protect islet ß cell injury in vitro. However, the efficacy of FTZ on ß cell regeneration in vivo and the involved anti-diabetic mechanism remains unknown. AIM OF THE STUDY: We aim to investigate the effects of FTZ as a good remedy for islet protection and ß cell regeneration, and to reveal the underlying mechanism. MATERIALS AND METHODS: C57BL/6 mice were fed with high-fat diet for 3 weeks and then intraperitoneally injected with streptozotocin (90 mg/kg/d × 1 d) to establish type 2 diabetes (T2D) models. Mice in each group were divided into three batches that sacrificed after 3, 7 and 28 days of FTZ administration. Body weight, blood glucose, and oral glucose tolerance test were measured at indicated time points. Fasting insulin was determined by enzyme-linked immunosorbent assay (ELISA) kit. Neonatal ß cell was assessed by insulin & PCNA double immunofluorescence staining, and the underlying mechanisms related to ß cell regeneration were further performed by hematoxylin-eosin staining, insulin & glucagon double immunofluorescence staining and Western blot. RESULTS: FTZ and metformin can significantly help with the symptoms of DM, such as alleviating weight loss, reducing blood glucose, improving the level of insulin in vivo, and relieving insulin resistance, suggesting FTZ and metformin treatment maintained the normal morphological function of islet. Notably, ß cell regeneration, which is indicated by insulin and PCNA double-positive cells, was promoted by FTZ, whereas few neonatal ß cells were observed in metformin group. Hematoxylin-eosin staining, and its quantification results showed that FTZ effectively prevented the invasion of inflammatory cells into the islets in diabetic mice. Most ß cells in the islets of diabetic model mice were devoid, and the islets were almost all α cells, while the diabetic mice administered FTZ could still maintain about half of the ß cells in the islet. Furthermore, FTZ upregulated the expression of critical transcription factors during ß cell development and maturation (such as PDX-1, MAFA and NGN3) in diabetic mice. CONCLUSIONS: FTZ can alleviate diabetes symptoms and promote ß cell regeneration in diabetic mice. Moreover, FTZ promotes ß cell regeneration by preserving islet (resisting inflammatory cells invading islets), maintaining the number of ß cells in islets, and increasing the expression of PDX-1, MAFA and NGN3.

Chinese medicine Fufang Zhenzhu Tiaozhi capsule ameliorates coronary atherosclerosis in diabetes mellitus-related coronary heart disease minipigs.

BACKGROUND: Diabetes mellitus-related coronary heart disease (DM-CHD) is the most common cause of death in diabetic patients. Various studies have shown that Chinese medicine Fufang-Zhenzhu-Tiaozhi capsule (FTZ) has therapeutic effects on cardiovascular diseases. More research is required to determine the mechanism of FTZ protection against coronary atherosclerosis. OBJECTIVE: To investigate the unique mechanism of FTZ in treatment of DM-CHD minipigs with coronary atherosclerosis. METHODS: High-fat/high-sucrose/high-cholesterol diet combined with streptozotocin and coronary balloon injury were used to induce DM-CHD minipig model, which was then randomly divided into: DM-CHD model, DM-CHD treated with FTZ or positive drug (Metformin + Atorvastatin, M+A). After twenty-two weeks, ultrasonography, electrocardiography, and image detection were employed to detect cardiac functions and assess coronary artery stenosis and plaque. Human umbilical vein endothelial cells (HUVECs) were treated high glucose or/and FTZ. Pigs tissues and treated-cells were collected for further testing. RESULTS: In DM-CHD minipigs, FTZ treatment significantly reduced disordered glycolipid metabolism similar as M+A administration. FTZ and M+A also alleviated coronary stenosis and myocardial injury. In addition, IκB and NF-κB phosphorylation levels, as well as the protein levels of IL-1ß, Bax, cleave-Caspase 3, Bcl-2, and α-SMA were dramatically increased in the DM-CHD coronary artery, whereas CD31 and VE-cadherin expressions were decreased. Similar to M+A, FTZ reversed these protein levels in the DM-CHD coronary artery. Furthermore, FTZ ameliorated the damage and high migration activity of HUVECs induced by high glucose. CONCLUSIONS: FTZ improves coronary atherosclerosis through modulating inflammation, alleviating apoptosis, and inhibiting EndMT of coronary artery to protects against DM-CHD.

Astragalus mongholicus powder, a traditional Chinese medicine formula ameliorate type 2 diabetes by regulating adipoinsular axis in diabetic mice.

The global morbidity of obesity and type 2 diabetes mellitus (T2DM) has dramatically increased. Insulin resistance is the most important pathogenesis and therapeutic target of T2DM. The traditional Chinese medicine formula Astragalus mongholicus powder (APF), consists of Astragalus mongholicus Bunge [Fabaceae], Pueraria montana (Lour.) Merr. [Fabaceae], and Morus alba L. [Moraceae] has a long history to be used to treat diabetes in ancient China. This work aims to investigate the effects of APF on diabetic mice and its underlying mechanism. Diabetic mice were induced by High-fat-diet (HFD) and streptozotocin (STZ). The body weight of mice and their plasma levels of glucose, insulin, leptin and lipids were examined. Reverse transcription-polymerase chain reaction, histology, and Western blot analysis were performed to validate the effects of APF on diabetic mice and investigate the underlying mechanism. APF reduced hyperglycemia, hyperinsulinemia, and hyerleptinemia and attenuate the progression of obesity and non-alcoholic fatty liver disease (NAFLD). However, these effects disappeared in leptin deficient ob/ob diabetic mice and STZ-induced insulin deficient type 1 diabetic mice. Destruction of either these hormones would abolish the therapeutic effects of APF. In addition, APF inhibited the protein expression of PTP1B suppressing insulin-leptin sensitivity, the gluconeogenic gene PEPCK, and the adipogenic gene FAS. Therefore, insulin-leptin sensitivity was normalized, and the gluconeogenic and adipogenic genes were suppressed. In conclusion, APF attenuated obesity, NAFLD, and T2DM by regulating the balance of adipoinsular axis in STZ + HFD induced T2DM mice.

Traditional Chinese Medicine Fufang-Zhenzhu-Tiaozhi capsule prevents renal injury in diabetic minipigs with coronary heart disease.

BACKGROUND: Renal injury is one of the common microvascular complications of diabetes, known as diabetic kidney disease (DKD) seriously threatening human health. Previous research has reported that the Chinese Medicine Fufang-Zhenzhu-Tiaozhi (FTZ) capsule protected myocardia from injury in diabetic minipigs with coronary heart disease (DM-CHD). And we found significant renal injury in the minipigs. Therefore, we further investigated whether FTZ prevents renal injury of DM-CHD minipig and H2O2-induced oxidative injury of HK-2 cells. METHODS: DM-CHD model was established by streptozotocin injection, high fat/high-sucrose/high-cholesterol diet combined with balloon injury in the coronary artery. Blood lipid profile, fasting blood glucose (FBG), and SOD were measured with kits. The levels of blood urea nitrogen (BUN), serum creatinine (Scr), urine trace albumin (UALB), urine creatinine (UCR) (calculate UACR), cystatin (Cys-C), and ß-microglobulin (ß-MG) were measured by ELISA kits to evaluate renal function. TUNEL assay was performed to observe the apoptosis. qPCR was used to detect the mRNA expression levels of HO-1, NQO1, and SOD in kidney tissue. The protein expressions of Nrf2, HO-1, NQO1, Bax, Bcl-2, and Caspase 3 in the kidney tissue and HK-2 cells were detected by western blot. Meanwhile, HK-2 cells were induced by H2O2 to establish an oxidative stress injury model to verify the protective effect and mechanisms of FTZ. RESULTS: In DM-CHD minipigs, blood lipid profile and FBG were elevated significantly, and the renal function was decreased with the increase of BUN, Scr, UACR, Cys-c, and ß-MG. A large number of inflammatory and apoptotic cells in the kidney were observed accompanied with lower levels of SOD, Bcl-2, Nrf2, HO-1, and NQO1, but high levels of Bax and Cleaved-caspase 3. FTZ alleviated glucose-lipid metabolic disorders and the pathological morphology of the kidney. The renal function was improved and the apoptotic cells were reduced by FTZ administration. FTZ could also enhance the levels of SOD, Nrf2, HO-1, and NQO1 proteins to promote antioxidant effect, down-regulate the expression of Bax and Caspase3, as well as up-regulate the expression of Bcl-2 to inhibit cell apoptosis in the kidney tissue and HK-2 cells. CONCLUSIONS: We concluded that FTZ prevents renal injury of DM-CHD through activating anti-oxidative capacity to reduce apoptosis and inhibiting inflammation, which may be a new candidate for DKD treatment.

FTZ protects against cardiac hypertrophy and oxidative injury via microRNA-214 / SIRT3 signaling pathway.

BACKGROUND: Despite the fact that the initial hypertrophic response to ventricular pressure overload is thought to be compensatory, prolonged stress often leads to heart failure. Previous studies have shown that the Fufang-Zhenzhu-Tiaozhi (FTZ) formula is beneficial for the treatment of dyslipidemia and hyperglycemia. However, the effects of FTZ on cardiac hypertrophy remain unclear. OBJECTIVE: The aim of this study is to evaluate the protective effects of FTZ on cardiac hypertrophy and determine the underlying mechanisms. METHODS: TAC was utilized to establish a cardiac hypertrophy animal model, and FTZ was given via gavage for four weeks. Next, echocardiographic measurements were made. The morphology of mouse cardiomyocytes was examined using H&E and WGA staining. In vitro, the neonatal cardiomyocytes were stimulated with angiotensin Ⅱ (Ang Ⅱ). In addition to measuring the size of cardiomyocytes, qRT-PCR and western blotting were conducted to measure cardiac stress markers and pathway. RESULTS: According to our findings, FTZ alleviated cardiac hypertrophy in mice and cell models. Furthermore, expression of miR-214 was down-regulated following FTZ, whereas the effect of FTZ therapy was reversed using miR-214 transfection. Furthermore, the expression of Sirtuin 3 (SIRT3) was decreased in Ang Ⅱ-induced oxidative damage, which was associated with a reduction in SOD-1, GPX1, and HO-1 and an increase in MDA, while SIRT3 expression was restored following FTZ treatment. CONCLUSIONS: Collectively, these findings indicate that FTZ is a protective factor for cardiac hypertrophy due to its regulation of the miR-214-SIRT3 axis, which suggests that FTZ may be a therapeutic target for cardiac hypertrophy.

FTZ Ameliorates Diabetic Cardiomyopathy by Inhibiting Inflammation and Cardiac Fibrosis in the Streptozotocin-Induced Model.

BACKGROUND: The pathogenesis and clinical features of diabetic cardiomyopathy (DCM) have been well studied in the past decade; however, effective approaches to prevent and treat this disease are limited. Fufang Zhenzhu Tiaozhi (FTZ) formula, a traditional Chinese prescription, is habitually used to treat dyslipidemia and diabetes. Recently, several studies have reported the therapeutic effects of FTZ on cardiovascular diseases. However, the effects of FTZ on DCM have not yet been fully elucidated. This study investigated the effects of FTZ on DCM and determined the mechanisms underlying its efficacy. METHODS: Diabetes was induced in mice by intraperitoneal injection of streptozotocin; the mice were randomly divided into a control group (Con), diabetes group (DCM), and diabetes-treated with FTZ (DCM + FTZ). Myocardial structural alterations, fibrosis biomarkers, and inflammation were observed. Besides, the potential targets and their related signaling pathways were analyzed using network pharmacology and further verified by Western blot. RESULTS: Diabetic mice showed significant body weight loss, hyperglycemia, and excessive collagen content in the cardiac tissue, while serum and myocardial inflammatory factors significantly increased. Nerveless, treatment with FTZ for 1 month significantly improved body weight, attenuated hyperglycemia, and alleviated diabetes-associated myocardial structure and function abnormalities. Furthermore, the serum levels of interleukin 12 (IL-12) and chemokine (C-C motif) ligand 2 (CCL2) as well as the mRNA levels of cardiac IL-12, IL-6, and C-C motif chemokine receptor 2 (Ccr2) reduced after FTZ treatment. Additionally, a total of 67 active compounds and 76 potential targets related to DCM were analyzed. Pathway and functional enrichment analyses showed that FTZ mainly regulates inflammation-related pathways, including MAPK and PI3K-AKT signaling pathways. Further investigation revealed that the activities of STAT3, AKT, and ERK were augmented in diabetic hearts but decreased in FTZ-treated cardiac tissues. CONCLUSION: Our results suggest that FTZ exhibits therapeutic properties against DCM by ameliorating hyperglycemia-induced inflammation and fibrosis via at least partial inhibition of AKT, ERK, and STAT3 signaling pathways.

Long-Term Effect of Zhenzhu Tiaozhi Capsule (FTZ) on Hyperlipidemia: 2-Year Results from a Retrospective Study Using Electronic Medical Records.

The objective of this work was to study the lipid profile (LDL, TC, TG, and HDL) over 2 years in patients with hyperlipidemia in a real-world clinical setting and to describe the dynamical trajectory of lipid profile change in response to lipid-lowering treatment (Zhenzhu Tiaozhi capsule (FTZ) vs. general lipid-lowering treatment, i.e., statins, fibrates, and Xuezhikang). We conducted a retrospective study that included people aged ≥18 years with hyperlipidemia that initiated lipid-lowering treatment between January 2010 and December 2020. Demographic, diagnosis, and laboratory data were retrieved from hospital's electronic medical records, including hospital information system (HIS) and the laboratory information system (LIS). Follow-up trajectories of lipid profile were plotted in a generalized additive mixed model (GAMM) with smooth splines. A total of 839 patients with hyperlipidemia were included. Within 2 years, LDL, TC, and TG descended steadily and gently in the FTZ group (N = 99), while the general lipid-lowering treatment (N = 740) shortly improved LDL, TC, and TG before 11 weeks and was no longer present around 30 weeks. After 30 weeks, the trajectory of LDL, TC, and TG fluctuated up and down. Also, for HDL, a similar trajectory was observed before 40 weeks between 2 groups, but the FTZ group showed an increasing trend after 40 weeks, while a similar trend was not seen in the general lipid-lowering group. In this study, FTZ was shown to have similar long-term effectiveness as an alternative lipid-lowering treatment to the general lipid-lowering treatment. The findings of this study provide observational evidence for further studies of FTZ, but more prospective studies are needed to determine the impacts of FTZ on lipid profile.

FTZ attenuates liver steatosis and fibrosis in the minipigs with type 2 diabetes by regulating the AMPK signaling pathway.

Fufang Zhenzhu Tiaozhi formula (FTZ), a preparation of Chinese herbal medicine, has various pharmacological properties, such as hypoglycemic, hypolipidemic, anticoagulant, and anti-inflammatory activities. Hepatocyte apoptosis is a marker of nonalcoholic steatohepatitis (NASH) and contributes to liver injury, fibrosis, and inflammation. Given the multiple effects of FTZ, we investigated whether FTZ can be a therapeutic agent for NASH and its mechanism. In the present study, we observed that FTZ treatment had an obviously favorable influence on hepatic steatosis and fibrosis in the histopathologic features of type 2 diabetes mellitus (T2DM) and coronary heart disease (CHD) with NASH minipigs. In addition, immunohistochemical analysis showed increased expression of the fibrotic marker α-smooth muscle actin (α-SMA), and a TUNEL assay revealed increased apoptotic positive hepatic cells in the liver tissues of the model group. Furthermore, FTZ administration reduced the increased expression of α-SMA, and FTZ inhibited apoptosis by affecting Bcl-2/Bax and cleaved caspase-3 expression. Mechanistically, our data suggested that FTZ treatment attenuated hepatic steatosis and fibrosis via the adenosine monophosphate-activated protein kinase (AMPK) pathway. In vitro studies showed that FTZ also attenuated intracellular lipid accumulation in HepG2 cells exposed to palmitic acid (PA) and oleic acid (OA). FTZ upregulated the expression levels of P-AMPK and BCL-2 and downregulated BAX. The changes induced by FTZ were reversed by Compound C, an inhibitor of AMPK. In conclusion, FTZ attenuated NASH by ameliorating steatosis and hepatocyte apoptosis, which is attributable to the regulation of the AMPK pathway.

The Efficacy and Safety of Chinese Medicine Fufang Zhenzhu Tiaozhi Capsule (FTZ) in the Treatment of Diabetic Coronary Heart Disease: Study Protocol for Multicenter, Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

BACKGROUND: Diabetic coronary heart disease (DCHD), the main macrovascular complication of type 2 diabetes mellitus (T2DM), is greatly harmful to T2DM patients. Traditional Chinese medicine (TCM) is an alternative and effective therapy to delay the development of macrovascular diseases, but the existing evidence of its efficacy and safety is insufficient. The aim of this multicenter, randomized, double-blind, placebo-controlled trial is to evaluate the efficacy and safety of Chinese Medicine Fufang Zhenzhu Tiaozhi capsule (FTZ) in treating DCHD. PATIENTS AND METHODS: This study includes a 2-week run-in, 52-week treatment, and 52-week post-treatment follow-up. A total of 160 participants will be recruited and randomized into two groups. The treatment group will receive FTZ and basic treatment, while the control group will receive the placebo and basic treatment. The primary outcome is the combined outcome including the major adverse cardiovascular events, coronary restenosis, and unplanned revascularization. The combined secondary outcomes include all-cause mortality, acute coronary syndrome, ischemic stroke, heart failure, unplanned re-hospitalization mainly caused by acute complications of diabetes, other thromboembolic events, and TCM symptom indicators. The safety outcomes and adverse events will also be evaluated in this trial. DISCUSSION: This trial evaluates the clinical effectiveness and safety of FTZ in patients with DCHD. The results are important to further explore the effectiveness of the comprehensive strategy "Tiao Gan Qi Shu Hua Zhuo" (modulating Gan, trigging key metabolic system to resolve pathogenic factors such as phlegm retention and dampness) in the prevention and control of glucolipid metabolic disorders (GLMD) including DCHD and T2DM. On the other hand, this study is the first trial of FTZ to observe cardiovascular outcomes through long-term follow-up after treatment of DCHD, which is of great value. TRIAL REGISTRATION: This trial was registered in the Chinese Clinical Trial Registry on April 07, 2019 (No. ChiCTR1900022345).

The traditional Chinese medicine formula Fufang-Zhenzhu-Tiaozhi protects myocardia from injury in diabetic minipigs with coronary heart disease.

BACKGROUND AND PURPOSE: Diabetes mellitus (DM) is a major risk factor for coronary heart disease (CHD). Previous research has reported that the Fufang-Zhenzhu-Tiaozhi (FTZ) formula has obvious effects on the treatment of dyslipidemia and hyperglycemia. In the present study, we intended to establish a convenient DM-CHD model in minipigs and investigated the protective effect of FTZ against myocardial injury and its mechanism. METHODS: The DM-CHD model was established by a high-fat/high-sucrose/high-cholesterol diet (HFSCD) combined with balloon injury in the coronary artery. Subsequently, sixteen Wuzhishan minipigs were assigned to three groups: control group, model group, and FTZ group. The model group and FTZ group were given a HFSCD, while the control group was given a normal diet (ND). FTZ was given with meals in the FTZ group. During this time, biochemical parameters, such as total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein (HDL-C), and fasting blood glucose (FBG), were measured by using testing kits. Insulin (INS) was determined by ELISA, and the homeostasis model assessment index of insulin resistance (HOMA-IR) was calculated to evaluate insulin resistance levels. After FTZ administration, the plasma levels of lactate dehydrogenase (LDH), creatine kinase isoenzyme MB (CK-MB), and cardiac troponin I (cTnI) were measured by using ELISA kits to evaluate myocardial injury. Coronary artery stenosis was analyzed by angiographic and HE staining. Myocardial ischemia was assayed with electrocardiogram (ECG). Moreover, cytokines, including interleukin-6 (IL-6), hypersensitive C-reactive protein (hs-CRP), and tumor necrosis factor-alpha (TNF-α), were measured by ELISA kits to assess inflammation. The myocardial tissue was collected, and the pathological morphology was observed by transmission electron microscopy (TEM), HE staining, and Masson staining. Western blots were used to detect the expression of PI3K, AKT, p-AKT, p-NF-κB, and NF-κB. RESULTS: A DM-CHD model in minipigs with glucose-lipid metabolism disorder, coronary artery incrassation and myocardial damage was successfully established through balloon injury in the coronary artery combined with HFSCD. FTZ effectively inhibited coronary artery incrassation and protected the myocardium against injury in DM-CHD minipigs. FTZ decreased proinflammatory cytokine levels and upregulated the protein expression of the PI3K/Akt pathway in the myocardium. CONCLUSIONS: A novel DM-CHD model in minipigs was successfully established through balloon injury in the coronary artery combined with HFSCD. FTZ has a protective effect against myocardial injury in DM-CHD by inhibiting inflammation and activating the PI3K/AKT signaling pathway.

Molecular mechanism of Fufang Zhenzhu Tiaozhi capsule in the treatment of type 2 diabetes mellitus with nonalcoholic fatty liver disease based on network pharmacology and validation in minipigs.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang Zhenzhu Tiaozhi formula (FTZ) of which a patented preparation of Chinese herbal medicine has been well documented with significant clinical curative effect for hyperglycemia and hyperlipidemia. Because of the complexity of the chemical constituents of Chinese herbal formulas, the holistic pharmacological mechanism of FTZ acting on type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD) remains unclear. AIM OF THE STUDY: To investigate the pharmacological efficacy and mechanism of FTZ in the treatment of T2DM accompanied by NAFLD. MATERIALS AND METHODS: Network pharmacology and validation in minipigs were used in this study. First, potential bioactive compounds of FTZ were identified by the traditional Chinese medicine system pharmacology technology platform (TCMSP). Then, targets of compounds were gathered using DrugBank, SwissTargetPrediction and TCMSP, while targets for T2DM and NAFLD were collected from CTD (compounds-targets-diseases network) and GeneCards. Common targets were defined as direct therapeutic targets acting on T2DM with NAFLD. In addition, crucial targets were chosen by the protein-protein interaction (PPI) network and contribution to compound-therapeutic targets in T2DM with the NAFLD network. Furthermore, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the metabolism-related signaling pathways affected by FTZ. Candidate patterns selected by network pharmacology were tested in the minipigs model of T2DM with NAFLD. Measurements of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), fasting insulin (FINS) and fasting blood glucose (FBG) in the blood and the expression levels of proteins, including PI3K-AKT and HIF-1α, in the livers of the minipigs were followed by the administration of FTZ. RESULTS: A total of 116 active compounds and 82 potential targets related to T2DM and NAFLD were found. Pathway and functional enrichment analysis showed that FTZ mainly regulates metabolism-related pathways, including PI3K-AKT, HIF-1α, TNFα and MAPK. Animal experiments showed that FTZ treatment significantly reduced the serum levels of TG, TC, LDL-C and FBG, increased serum levels of HDL-C, ameliorated systemic insulin resistance (IR), and attenuated liver damage in minipigs with T2DM and NAFLD. FTZ treatment has an obviously favorable influence on hepatic steatosis and liver lipid accumulation in the histopathologic features of HE, Oil red O staining, and electron microscopy. Mechanistically, FTZ improved liver metabolism by increasing the phosphorylation of PI3K-AKT and decreasing the expression of HIF-1α. CONCLUSION: Network pharmacology was supported by experimental studies, which indicated that FTZ has demonstrated therapeutic benefits in T2DM and NAFLD by regulating the PI3K-AKT and HIF-1α signaling pathways.

Cordycepin Ameliorates Nonalcoholic Steatohepatitis by Activation of the AMP-Activated Protein Kinase Signaling Pathway.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease, especially nonalcoholic steatohepatitis (NASH), has become a major cause of liver transplantation and liver-associated death. NASH is the hepatic manifestation of metabolic syndrome and is characterized by hepatic steatosis, inflammation, hepatocellular injury, and different degrees of fibrosis. However, there is no US Food and Drug Administration-approved medication to treat this devastating disease. Therapeutic activators of the AMP-activated protein kinase (AMPK) have been proposed as a potential treatment for metabolic diseases such as NASH. Cordycepin, a natural product isolated from the traditional Chinese medicine Cordyceps militaris, has recently emerged as a promising drug candidate for metabolic diseases. APPROACH AND RESULTS: We evaluated the effects of cordycepin on lipid storage in hepatocytes, inflammation, and fibrosis development in mice with NASH. Cordycepin attenuated lipid accumulation, inflammation, and lipotoxicity in hepatocytes subjected to metabolic stress. In addition, cordycepin treatment significantly and dose-dependently decreased the elevated levels of serum aminotransferases in mice with diet-induced NASH. Furthermore, cordycepin treatment significantly reduced hepatic triglyceride accumulation, inflammatory cell infiltration, and hepatic fibrosis in mice. In vitro and in vivo mechanistic studies revealed that a key mechanism linking the protective effects of cordycepin were AMPK phosphorylation-dependent, as indicated by the finding that treatment with the AMPK inhibitor Compound C abrogated cordycepin-induced hepatoprotection in hepatocytes and mice with NASH. CONCLUSION: Cordycepin exerts significant protective effects against hepatic steatosis, inflammation, liver injury, and fibrosis in mice under metabolic stress through activation of the AMPK signaling pathway. Cordycepin might be an AMPK activator that can be used for the treatment of NASH.

Chinese Medicine FTZ Recipe Protects against High-Glucose-Induced Beta Cell Injury through Alleviating Oxidative Stress.

OBJECTIVE: To investigate the effect of FTZ on high-glucose-induced oxidative stress and underlying mechanisms. METHODS: We used a ß cell dysfunction and diabetes model that was induced in rats fed a high-fat high-sugar diet (HFHSD) for 6 weeks and injected once with 35 mg/kg streptozocin (STZ). Then, 3 and 6 g/kg of FTZ were administered by gavage for 8 weeks. In addition, an ex vivo model of oxidative stress was induced by stimulating INS-1 cells with 25 mmol/L glucose for 48 h. RESULT: The levels of fasting blood glucose (FBG) in diabetic model rats were obviously higher than those in the normal group; furthermore with reduced levels of ß cells, catalase (CAT), superoxide dismutase (SOD), and Bcl-2 increased lipid peroxide malondialdehyde (MDA) and caspase-3 in the pancreatic tissue of the diabetic model rats. Afterward, the cells were incubated with FTZ-containing serum and edaravone. The 25 mmol/L glucose-induced SOD reduction increased MDA and intracellular ROS. The protein expression level of Mn-SOD and CAT in the model group decreased significantly compared with that in the control group. CONCLUSION: FTZ treatment significantly improved the alteration in the level of SOD, CAT, Bcl-2, caspase-3, and MDA coupled with ß cell dysfunction in diabetic rats. Oxidative stress in INS-1 cells was closely associated with a higher rate of apoptosis, increased production of ROS and MDA, enhanced Bax expression, and caspase-3, -9 activities and markedly decreased protein expression of Mn-SOD and CAT. FTZ-containing serum incubation notably reversed the high-glucose-evoked increase in cell apoptosis, production of ROS and MDA, and Bax protein levels. Furthermore, FTZ stimulation upregulated the expression levels of several genes, including Mn-SOD, CAT, and Bcl-2/Bcl-xl. In addition, FTZ decreased the intracellular activity of caspase-3, -9 in INS-1 cells. FTZ protected ß-cells from oxidative stress induced by high glucose in vivo and in vitro. The beneficial effect of FTZ was closely associated with a decrease in the activity of caspase-3, -9 and intracellular production of ROS, MDA, and Bax coupled with an increase in the expression of Mn-SOD, CAT, and Bcl-2/Bcl-xl.

Molecular characterization and expression of the MYND-ZF gene from Clonorchis sinensis.

The MYND-type zinc finger protein (MYND-ZF) is a large group of proteins containing the MYND domain which play an important role in protein-protein interactions. A cDNA clone encoding a novel MYND-ZF was isolated and identified from a Clonorchis sinensis (C. sinensis) adult cDNA library. The open reading frame of this novel cDNA sequence contains 1,440 base pairs with a putative protein of 479 amino acids showing a high homology with the MYND-ZF identified from other species. Recombinant CsMYND-ZF was expressed and purified from Escherichia coli BL21 (DE3). CsMYND-ZF transcripts were detected in the cDNA of adult worms and metacercariae but not in eggs of C. sinensis. Immunohistochemistry results revealed that CsMYND-ZF was deposited at the tegument of adult worms and metacercariae C. sinensis using anti-recombinant CsMYND-ZF serum. These findings may contribute to the development of a reliable diagnostic method.

Evaluation of the adjuvant properties of Astragalus membranaceus and Scutellaria baicalensis GEORGI in the immune protection induced by UV-attenuated Toxoplasma gondii in mouse models.

Human vaccines are not available and current anti-toxoplasma treatment is disappointing. To investigate the possible adjuvant effect of aqueous extracts obtained from medicinal herbs of Astragalus membranaceus (Am) and Scutellaria baicalensis GEORGI (Sb) on the immune response to Toxoplasma gondii in the mouse models induced by ultraviolet (UV)-attenuated T. gondii, this paper studies the possible vaccination strategies to help combat infections with Toxoplasma and looking towards developing new vaccine and approaches. We used UV-attenuated T. gondii (UV-T.g) of RH strain as a vaccine and the extracts of Am (AmE) and Sb (SbE) as adjuvant. Mice were infected by intraperitoneal (i.p.) injection of 10(2) RH tachyzoites alone (infected controls), infected and treated with AmE (T.g+AmE) and SbE (T.g+SbE), respectively; and mice immunized i.p. with UV-T.g alone, UV-T.g co-administrated with AmE (UV-T.g+AmE) or SbE (UV-T.g+SbE), and then challenged with T.g, respectively. The animal survival time, parasite burden in peritoneal lavage fluids, liver histopathological analysis, and levels of serum antibodies among the groups were compared after either infection or challenge. The results showed that, compared to infected controls, infected mice treated with AmE or SbE, or vaccinated mice and then challenged, had significantly prolonged survival time, decreased parasite burden, improved liver histopathological score, and increased Th1-type cellular immune response; furthermore, vaccinated mice co-administrated with AmE or SbE had even longer survival, lower parasite burden, lower liver histopathological score, and higher Th1 response after challenge. Our data demonstrated that the protective immunity of UV-attenuated T. gondii could be markedly enhanced by AmE or SbE co-administration, which suggests that both AmE and SbE may have the potential to be used as effective vaccine adjuvant.