Bazi Bushen ameliorates age-related energy metabolism dysregulation by targeting the IL-17/TNF inflammatory pathway associated with SASP.

BACKGROUND: Chronic inflammation and metabolic dysfunction are key features of systemic aging, closely associated with the development and progression of age-related metabolic diseases. Bazi Bushen (BZBS), a traditional Chinese medicine used to alleviate frailty, delays biological aging by modulating DNA methylation levels. However, the precise mechanism of its anti-aging effect remains unclear. In this study, we developed the Energy Expenditure Aging Index (EEAI) to estimate biological age. By integrating the EEAI with transcriptome analysis, we aimed to explore the impact of BZBS on age-related metabolic dysregulation and inflammation in naturally aging mice. METHODS: We conducted indirect calorimetry analysis on five groups of mice with different ages and utilized the data to construct EEAI. 12 -month-old C57BL/6 J mice were treated with BZBS or ß-Nicotinamide Mononucleotide (NMN) for 8 months. Micro-CT, Oil Red O staining, indirect calorimetry, RNA sequencing, bioinformatics analysis, and qRT-PCR were performed to investigate the regulatory effects of BZBS on energy metabolism, glycolipid metabolism, and inflammaging. RESULTS: The results revealed that BZBS treatment effectively reversed the age-related decline in energy expenditure and enhanced overall metabolism, as indicated by the aging index of energy expenditure derived from energy metabolism parameters across various ages. Subsequent investigations showed that BZBS reduced age-induced visceral fat accumulation and hepatic lipid droplet aggregation. Transcriptomic analysis of perirenal fat and liver indicated that BZBS effectively enhanced lipid metabolism pathways, such as the PPAR signaling pathway, fatty acid oxidation, and cholesterol metabolism, and improved glycolysis and mitochondrial respiration. Additionally, there was a significant improvement in inhibiting the inflammation-related arachidonic acid-linoleic acid metabolism pathway and restraining the IL-17 and TNF inflammatory pathways activated via senescence associated secretory phenotype (SASP). CONCLUSIONS: BZBS has the potential to alleviate inflammation in metabolic organs of naturally aged mice and maintain metabolic homeostasis. This study presents novel clinical therapeutic approaches for the prevention and treatment of age-related metabolic diseases.

Angelica keiskei water extract Mitigates Age-Associated Physiological Decline in Mice.

OBJECTIVE: Angelica keiskei is a medicinal and edible plant that has been reported to possess potent antioxidant properties in several in vitro models, but its effectiveness on naturally aging organisms is still lacking. This study explores the antioxidant and health-promoting effects of Angelica keiskei in naturally aging mice. METHODS: We treated 48-week-old mice with Angelica keiskei water extract (AKWE) 30 days, and measured indicators related to aging and antioxidants. In addition, we conducted network pharmacology analysis, component-target molecular docking, real-time PCR, and MTS assays to investigate relevant factors. RESULTS: The results indicated that administration of AKWE to mice led to decrease blood glucose levels, improve muscle fiber structure, muscle strength, gait stability, and increase levels of glutathione and superoxide dismutase in serum. Additionally, it decreased pigmentation of the heart tissues. Angelica keiskei combats oxidative stress by regulating multiple redox signaling pathways, and its ingredients Coumarin and Flavonoids have the potential to bind to SIRT3 and SIRT5. CONCLUSIONS: Our findings indicated the potential of Angelica keiskei as a safe and effective dietary supplement to combat aging and revealed the broad prospects of medicinal and edible plants for addressing aging and age-related chronic diseases.

Tongxinluo Activates PI3K/AKT Signaling Pathway to Inhibit Endothelial Mesenchymal Transition and Attenuate Myocardial Fibrosis after Ischemia-Reperfusion in Mice.

OBJECTIVE: To investigate the potential role of Tongxinluo (TXL) in attenuating myocardial fibrosis after myocardial ischemia-reperfusion injury (MIRI) in mice. METHODS: A MIRI mouse model was established by left anterior descending coronary artery ligation for 45 min. According to a random number table, 66 mice were randomly divided into 6 groups (n=11 per group): the sham group, the model group, the LY-294002 group, the TXL group, the TXL+LY-294002 group and the benazepril (BNPL) group. The day after modeling, TXL and BNPL were administered by gavage. Intraperitoneal injection of LY-294002 was performed twice a week for 4 consecutive weeks. Echocardiography was used to measure cardiac function in mice. Masson staining was used to evaluate the degree of myocardial fibrosis in mice. Qualitative and quantitative analysis of endothelial mesenchymal transition (EndMT) after MIRI was performed by immunohistochemistry, immunofluorescence staining and flow cytometry, respectively. The protein expressions of platelet endothelial cell adhesion molecule-1 (CD31), α-smoth muscle actin (α-SMA), phosphatidylinositol-3-kinase (PI3K) and phospho protein kinase B (p-AKT) were assessed using Western blot. RESULTS: TXL improved cardiac function in MIRI mice, reduced the degree of myocardial fibrosis, increased the expression of CD31 and inhibited the expression of α-SMA, thus inhibited the occurrence of EndMT (P<0.05 or P<0.01). TXL significantly increased the protein expressions of PI3K and p-AKT (P<0.05 or P<0.01). There was no significant difference between TXL and BNPL group (P>0.05). In addition, the use of the PI3K/AKT pathway-specific inhibitor LY-294002 to block this pathway and combination with TXL intervention, eliminated the protective effect of TXL, further supporting the protective effect of TXL. CONCLUSION: TXL activated the PI3K/AKT signaling pathway to inhibit EndMT and attenuated myocardial fibrosis after MIRI in mice.

Antidepressant-like effects of Jieyu Chufan capsules in the olfactory bulbectomy rat model.

The olfactory bulbectomy (OBX) animal model of depression reproduces the behavioral and neurochemical changes observed in depressed patients. We assessed the therapeutic effects of the Jieyu Chufan (JYCF) capsule on OBX rats. JYCF ameliorated the hedonic and anxiety-like behavior of OBX rats and attenuated the cortical and hippocampal damage. JYCF enhanced the expression of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), fibroblast growth factor 2 (FGF2), and adiponectin (ADPN) in the cortex and hippocampus of OBX rats. JYCF also reduced cortisol levels and restored the levels of excitatory neurotransmitters, such as 5-hydroxytryptamine (5-HT), acetylcholine (ACH), and glutamic acid (Glu), in the brain tissue of OBX rats. Our results suggest that JYCF preserves the synaptic structure by increasing the levels of synaptophysin (SYN) and postsynaptic density protein 95 (PSD95) and alleviates the histological alterations of brain tissue by activating AKT/PKA-CREB-BDNF pathways, and by upregulating ADPN and FGF2 expression in OBX rats. JYCF exerts multiple therapeutic effects on depression, including modulating neurotransmitters, repairing neuronal damage, and maintaining synaptic integrity. These findings support the potential of JYCF as a novel antidepressant agent with therapeutic effects on depression and related neurological disorders.

Acupuncture Treatment in a Mouse Model of Chronic Hypoxia-Induced Cognitive Dysfunction.

The treatment of central nervous disorders has consistently posed significant challenges to the medical field. Acupuncture, a non-pharmacological practice rooted in traditional Chinese medicine, entails the insertion of fine needles into precise points on the body and is commonly employed for the management of diverse conditions. Recently, acupuncture has emerged as a promising therapeutic intervention for a range of neurological diseases, including anxiety and respiratory disorders. However, the potential of acupuncture in treating cognitive dysfunction induced by chronic hypoxia has not yet been explored. This paper presents a comprehensive protocol for establishing a mouse model of chronic hypoxia-induced cognitive impairment, administering mild anesthesia, performing acupuncture treatment, and assessing behavioral changes and memory abilities using open field tests and water mazes. The step-by-step protocol provides detailed instructions on accurately locating and positioning acupoints and needles for cognitive improvement. By employing this protocol, researchers can conduct systematic studies to thoroughly evaluate the therapeutic potential of acupuncture for cognitive dysfunction.

Therapeutic potential of Lianhua Qingke in airway mucus hypersecretion of acute exacerbation of chronic obstructive pulmonary disease.

BACKGROUND: Lianhua Qingke (LHQK) is an effective traditional Chinese medicine used for treating acute tracheobronchitis. In this study, we evaluated the effectiveness of LHQK in managing airway mucus hypersecretion in the acute exacerbation of chronic obstructive pulmonary disease (AECOPD). METHODS: The AECOPD model was established by subjecting male Wistar rats to 12 weeks of cigarette smoke (CS) exposure (80 cigarettes/day, 5 days/week for 12 weeks) and intratracheal lipopolysaccharide (LPS) exposure (200 µg, on days 1, 14, and 84). The rats were divided into six groups: control (room air exposure), model (CS + LPS exposure), LHQK (LHQK-L, LHQK-M, and LHQK-H), and a positive control group (Ambroxol). H&E staining, and AB-PAS staining were used to evaluate lung tissue pathology, inflammatory responses, and goblet cell hyperplasia. RT-qPCR, immunohistochemistry, immunofluorescence and ELISA were utilized to analyze the transcription, expression and secretion of proteins related to mucus production in vivo and in the human airway epithelial cell line NCI-H292 in vitro. To predict and screen the active ingredients of LHQK, network pharmacology analysis and NF-κB reporter system analysis were employed. RESULTS: LHQK treatment could ameliorate AECOPD-triggered pulmonary structure damage, inflammatory cell infiltration, and pro-inflammatory cytokine production. AB-PAS and immunofluorescence staining with CCSP and Muc5ac antibodies showed that LHQK reduced goblet cell hyperplasia, probably by inhibiting the transdifferentiation of Club cells into goblet cells. RT-qPCR and immunohistochemistry of Muc5ac and APQ5 showed that LHQK modulated mucus homeostasis by suppressing Muc5ac transcription and hypersecretion in vivo and in vitro, and maintaining the balance between Muc5ac and AQP5 expression. Network pharmacology analysis and NF-κB luciferase reporter system analysis provided insights into the active ingredients of LHQK that may help control airway mucus hypersecretion and regulate inflammation. CONCLUSION: LHQK demonstrated therapeutic effects in AECOPD by reducing inflammation, suppressing goblet cell hyperplasia, preventing Club cell transdifferentiation, reducing Muc5ac hypersecretion, and modulating airway mucus homeostasis. These findings support the clinical use of LHQK as a potential treatment for AECOPD.

Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats.

This study aims to show the estrogen-like effect of Bazi Bushen capsule (BZBS), a Chinese herbal compound, in ovariectomized mice. Female Sprague-Dawley (SD) rats were randomly divided into six groups: a sham-operated group, a model group (OVX), a progynova group, and BZBS groups (1, 2, and 4 d/kg/d). An ovariectomy was performed on all rats except those in the sham-operated group. Micro-computed tomography (micro-CT) scanning, hematoxylin and eosin (H&E) staining, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) detection were performed after 4 months of BZBS treatment. As a result, compared with the OVX group, rats treated with BZBS showed an increased number and area of trabecular bone and bone marrow cells, and a decreased number of adipose cells. The bone volume, trabecular number, and trabecular thickness of the right tibia in the medication groups increased and the trabecular space decreased. The 17ß-estradiol and serum calcium levels in the medication groups were elevated, but the levels of serum phosphorus, sclerostin, ß-CTX, and TRACP-5b were decreased. In the medication groups, the RANKL and sclerostin levels were decreased, while the osteoprotegerin (OPG) level was increased. In conclusion, this protocol systematically evaluated the therapeutic effects and potential molecular mechanisms of Chinese herbal compounds in ovariectomized rats with a variety of techniques.

Shensong Yangxin Capsule Reduces the Susceptibility of Arrhythmia in db/db Mice via Inhibiting the Inflammatory Response Induced by Endothelium Dysfunction.

Purpose: The aim of our study was to investigate the mechanism by which the Chinese compound Shensong Yangxin Capsule (SSYX) reduces susceptibility to arrhythmia in db/db mice. Methods: The db/db mice without drug treatment served as the model group. Other-treated db/db mice were administered SSYX for 8 weeks. Electrocardiogram (ECG), electrical mapping, pathological changes, immunofluorescence staining, real-time quantitative PCR, and Western blot analyses were then conducted. Results: SSYX decreased arrhythmia susceptibility and shortened the abnormal ECG parameters of db/db mice. Meanwhile, SSYX restored irregular conduction direction and shortened the conduction time of the isolated heart. HE and Masson staining showed that SSYX alleviated inflammatory infiltration and collagen fiber deposition. Western blot showed that SSYX decreased the protein expression of ICAM-1, VCAM-1, and MCP-1 and increased the protein expression of occludin, ZO-1, eNOS, and Cx43. SSYX also increased the content of NO, decreased ET-1, TNF-α, IL-1ß, IL-6, MCP-1, and CCR-2 mRNA expression, and increased Kv 4.2, Kv 4.3, Cav 1.2, and Nav 1.5 mRNA expression. Furthermore, SSYX decreased the fluorescence intensity of F4/80 and iNOS, increased the fluorescence intensity of CD31 and eNOS, and improved the Cx43 and α-actinin connection structure in cardiac tissues. The above therapeutic effects of SSYX were inhibited by L-NAME. Conclusion: SSYX reduced the susceptibility of db/db mice to arrhythmia by inhibiting the inflammatory response and macrophage polarization, and this effect of SSYX occurred through protection of endothelial cell function.

Bazi Bushen mitigates epigenetic aging and extends healthspan in naturally aging mice.

Bazi Bushen (BZBS), a traditional Chinese medicine, has been proven effective in the treatment of age-related disease in mouse models. However, whether its therapeutic effects are due to antiaging mechanism has not yet been explored. In the present study, we investigated the antiaging effects of BZBS in naturally aging mice by using behavioral tests, liver DNA methylome sequencing, methylation age estimation, and frailty index assessment. The methylome analysis revealed a decrease of mCpG levels in the aged mouse liver. BZBS treatment tended to restore age-associated methylation decline and prune the methylation pattern toward that of young mice. More importantly, BZBS significantly rejuvenated methylation age of the aged mice, which was computed by an upgraded DNA methylation clock. These results were consistent with enhanced memory and muscular endurance, as well as decreased frailty score and liver pathological changes. KEGG analysis together with aging-related database screening identified methylation-targeted pathways upon BZBS treatment, including oxidative stress, DNA repair, MAPK signaling, and inflammation. Upregulation of key effectors and their downstream effects on elevating Sod2 expression and diminishing DNA damage were further investigated. Finally, in vitro experiments with senescent HUVECs proved a direct effect of BZBS extracts on the regulation of methylation enzymes during cellular aging. In summary, our work has revealed for the first time the antiaging effects of BZBS by slowing the methylation aging. These results suggest that BZBS might have great potential to extend healthspan and also explored the mechanism of BZBS action in the treatment of age-related diseases.

Sophoridine manifests as a leading compound for anti-arrhythmia with multiple ion-channel blocking effects.

BACKGROUND: Sophoridine (SR) has shown the potential to be an antiarrhythmic agent. However, SR's electrophysiological properties and druggability research are relatively inadequate, which limits the development of SR as an antiarrhythmic candidate. PURPOSE: To facilitate the development process of SR as an antiarrhythmic candidate, we performed integrated studies on the electrophysiological properties of SR in vitro and ex vivo to gain more comprehensive insights into the multi-ion channel blocking effects of SR, which provided the foundation for the further drugability studies in antiarrhythmic and safety studies. Firstly, SR's electrophysiological properties and antiarrhythmic potentials were recorded and assessed at the cell and tissue levels by comprehensively integrating the patch clamp with the Electrical and Optical Mapping systems. Subsequently, the antiarrhythmic effects of SR were validated by aconitine and ouabain-induced arrhythmia in vivo. Finally, the safety of SR as an antiarrhythmic candidate compound was evaluated based on the guidelines of the Comprehensive in Vitro Proarrhythmia Assay (CiPA). STUDY DESIGN: The antiarrhythmic effect of SR was evaluated at the in vitro, ex vivo, and in vivo levels. METHODS: Isolated primary cardiomyocytes and stable cell lines were prepared to explore the electrophysiologic properties of being a multiple ion-channel blocker in vitro by whole-cell patch clamp. Using electrical and optical mapping, the negative chronotropic effect of SR was determined in langendorff-perfused rat or guinea-pig hearts.The antiarrhythmic activity of SR was assessed by the ex vivo tachyarrhythmia models induced by left coronary artery ligation (LCAL) and isoproterenol (ISO). Canonical models of aconitine and ouabain-induced arrhythmia were used to verify the antiarrhythmic effects in vivo. Finally, the pro-arrhythmic risk of SR was detected in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hSCCMs) using a Microelectrode array (MEA). RESULTS: Single-cell patch assay validated the multiple ion-channel blockers of SR in transient outward current potassium currents (Ito), l-type calcium currents (ICa-l), and rapid activation delayed rectifier potassium currents (IKr). SR ex vivo depressed heart rates (HR) and ventricular conduction velocity (CV) and prolonged Q-T intervals in a concentration-dependent manner. Consistent with the changes in HRs, SR extended the active time of hearts and increased the action potential duration measured at 90% repolarization (APD90). SR could also significantly lengthen the onset time and curtail the duration of spontaneous ventricular tachycardia (VT) in the ex vivo arrhythmic model induced by LCAL. Meanwhile, SR could also significantly upregulate the programmed electrical stimulation (PES) frequency after the ISO challenge in forming electrical alternans and re-entrant excitation. Furthermore, SR exerted antiarrhythmic effects in the tachyarrhythmia models induced by aconitine and ouabain in vivo. Notably, the pro-arrhythmic risk of SR was shallow for a moderate inhibition of the human ether-à-go-go-related gene (hERG) channel. Moreover, SR prolonged field potential duration (FPDc) of hSCCMs in a concentration-dependent manner without early after depolarization (EAD) and arrhythmia occurrence. CONCLUSION: Our results indicated that SR manifested as a multiple ion-channel blocker in the electrophysiological properties and exerts antiarrhythmic effects ex vivo and in vivo. Meanwhile, due to the low pro-arrhythmic risk in the hERG inhibition assay and the induction of EAD, SR has great potential as a leading candidate in the treatment of ventricular tachyarrhythmia.

Bazi Bushen capsule attenuates cognitive deficits by inhibiting microglia activation and cellular senescence.

CONTEXT: Bazi Bushen capsule (BZBS) has anti-ageing properties and is effective in enhancing memory. OBJECTIVE: To find evidence supporting the mechanisms and biomarkers by which BZBS functions. MATERIALS AND METHODS: Male C57BL/6J mice were randomly divided into five groups: normal, ageing, ß-nicotinamide mononucleotide capsule (NMN), BZBS low-dose (LD-BZ) and BZBS high-dose (HD-BZ). The last four groups were subcutaneously injected with d-galactose (d-gal, 100 mg/kg/d) to induce the ageing process. At the same time, the LD-BZ, HD-BZ and NMN groups were intragastrically injected with BZBS (1 and 2 g/kg/d) and NMN (100 mg/kg/d) for treatment, respectively. After 60 days, the changes in overall ageing status, brain neuron morphology, expression of p16INK4a, proliferating cell nuclear antigen (PCNA), ionized calcium-binding adapter molecule 1 (Iba1), postsynaptic density protein 95 (PSD95), CD11b, Arg1, CD206, Trem2, Ym1 and Fizz1, and the senescence-associated secretory phenotype (SASP) factors were observed. RESULTS: Compared with the mice in the ageing group, the HD-BZ mice exhibited obvious improvements in strength, endurance, motor coordination, cognitive function and neuron injury. The results showed a decrease in p16INK4a, Iba1 and the upregulation of PCNA, PSD95 among brain proteins. The brain mRNA exhibited downregulation of Iba1 (p < 0.001), CD11b (p < 0.001), and upregulation of Arg1 (p < 0.01), CD206 (p < 0.05), Trem2 (p < 0.001), Ym1 (p < 0.01), Fizz1 (p < 0.05) and PSD95 (p < 0.01), as well as improvement of SASP factors. CONCLUSIONS: BZBS improves cognitive deficits via inhibition of cellular senescence and microglia activation. This study provides experimental evidence for the wide application of BZBS in clinical practice for cognitive deficits.

Jinlida Granules Reduce Obesity in db/db Mice by Activating Beige Adipocytes.

Recent studies indicate existence of beige adipocytes in adults. Upon activation, beige adipocytes burn energy for thermogenesis and contribute to regulation of energy balance. In this study, we have analyzed whether Jinlida granules (JLD) could activate beige adipocytes. JLD suspended in 0.5% carboxymethyl cellulose (CMC) was gavage fed to db/db mice at a daily dose of 3.8 g/kg. After 10 weeks, body weight, biochemical, and histological analyses were performed. In situ hybridization, immunofluorescence, and western blotting were conducted to test beige adipocyte activation in mice. X9 cells were induced with induction medium and maintenance medium containing 400 µg/mL of JLD. After completion of induction, cells were analyzed by Nile red staining, time polymerase chain reaction (PCR), western blotting, and immunofluorescence to understand the effect of JLD on the activation of beige adipocytes. A molecular docking method was used to preliminarily identify compounds in JLD, which hold the potential activation effect on uncoupling protein 1 (UCP1). JLD treatment significantly improved obesity in db/db mice. Biochemical results showed that JLD reduced blood glucose (GLU), triglyceride (TG), and low-density lipoprotein cholesterol (LDL) levels as well as liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in mice. Hematoxylin and eosin staining (H&E) showed that JLD reduced hepatocyte ballooning changes in the liver. Immunofluorescence showed that JLD increased the expression of the thermogenic protein, UCP1, in the beige adipose tissue of mice. JLD also increased the expression of UCP1 and inhibited the expression of miR-27a in X9 cells. Molecular docking results showed that epmedin B, epmedin C, icariin, puerarin, and salvianolic acid B had potential activation effects on UCP1. The results suggest that JLD may activate beige adipocytes by inhibiting miR-27a expression, thereby promoting thermogenesis in beige adipocytes. This study provides a new pharmacological basis for the clinical use of JLD.

Huogu injection alleviates SONFH by regulating adipogenic differentiation of BMSCs via targeting the miR-34c-5p/MDM4 pathway.

Although the precise pathogenesis of steroid-induced osteonecrosis of femoral head (SONFH) is not yet fully understood, evidence shows miRNAs-mediated posttranscription control directs the adipogenesis of bone marrow mesenchymal stem cells (BMSCs) and plays a pivotal role in the SONFH processes. Huogu injection formulated according to traditional Chinese medicine (TCM) theory has been used to treat SONFH by intra-articular injection. In this study, we asked whether the therapeutic effects of Huogu injection might depend on the inhibition of adipogenic differentiation of BMSCs, and if so, the pathway might be a therapeutic target to promote bone repair. Consequently, miR-34c-5p was upregulated in the dexamethasone (DEX)-treated BMSCs and might participate in the adipogenesis of BMSCs. TargetScan database and the luciferase reporter assay showed miR-34c-5p targeted on the MDM4 and negatively regulated its expression. Huogu injection in vitro inhibited the adipogenesis in the DEX-treated BMSCs by inhibiting the expression levels of PPARγ and C/EBPα, as well as reducing miR-34c-5p to prevent the degradation of MDM4. Moreover, miR-34c-5p mimic or MDM4 knockdown using shRNA neutralized the anti-adipogenesis of Huogu injection in BMSCs. In vivo, the results of X-ray imaging confirmed that Huogu injection alleviated the bone loss in rat SONFH. Consistent with results in vitro, Huogu injection reduced the lipid accumulation, removed the suppression of MDM4 by downregulating the expression of miR-34c-5p, and inhibited the expression of C/EBPα and PPARγ in bone tissues. When the lentivirus encoding miR-34c-5p was conducted by intra-articular injection, the overexpression of miR-34c-5p antagonized the therapeutic effects of Huogu injection. Our results underline the critical importance of the miR-34c-5p/MDM4 pathway in regulating the adipogenic outcome of BMSCs, suggesting the miR-34c-5p as a potentially effective therapeutic target in SONFH. These results further reinforce the potential of Huogu injection as an alternative approach in SONFH.

Jinlida granules ameliorate the high-fat-diet induced liver injury in mice by antagonising hepatocytes pyroptosis.

CONTEXT: Jinlida (JLD) as a traditional Chinese medicine formula has been used to treat type 2 diabetes mellitus (T2DM) and studies have shown its anti-obesity effect. OBJECTIVE: To investigate the therapeutic effects of JLD in a mouse model of non-alcoholic fatty liver (NAFL). MATERIALS AND METHODS: C57BL/6J mice were divided into three groups and fed a low-diet diet (LFD), high-fat diet (HFD), or HFD + JLD (3.8 g/kg) for 16 weeks, respectively. The free fatty acids-induced lipotoxicity in HepG2 cells were used to evaluate the anti-pyroptotic effects of JLD. The pharmacological effects of JLD on NAFL were investigated by pathological examination, intraperitoneal glucose and insulin tolerance tests, western blotting, and quantitative real-time PCR. RESULTS: In vivo studies showed that JLD ameliorated HFD-induced liver injury, significantly decreased body weight and enhanced insulin sensitivity and improved glucose tolerance. Furthermore, JLD suppressed both the mRNA expression of caspase-1 (1.58 vs. 2.90), IL-1ß (0.93 vs. 3.44) and IL-18 (1.34 vs. 1.60) and protein expression of NLRP3 (2.04 vs. 5.71), pro-caspase-1 (2.68 vs. 4.92) and IL-1ß (1.61 vs. 2.60). In vitro, JLD inhibited the formation of lipid droplets induced by 2 mM FFA (IC50 = 2.727 mM), reduced the protein expression of NLRP3 (0.74 vs. 2.27), caspase-1 (0.57 vs. 2.68), p20 (1.67 vs. 3.33), and IL-1ß (1.44 vs. 2.41), and lowered the ratio of p-IKB-α/IKB-α (0.47 vs. 2.19). CONCLUSION: JLD has a protective effect against NAFLD, which may be related to its anti-pyroptosis, suggesting that JLD has the potential as a novel agent in the treatment of NAFLD.

Tongxinluo prevents chronic obstructive pulmonary disease complicated with atherosclerosis by inhibiting ferroptosis and protecting against pulmonary microvascular barrier dysfunction.

Cardiovascular comorbidities are pervasive in chronic obstructive pulmonary disease (COPD) and often result in serious adverse cardiovascular events. Tongxinluo (TXL) has been clinically verified to treat atherosclerosis (AS), improve lung function and alleviate dyspnoea. The present study aimed to explore the effect of lung microvascular barrier dysfunction on AS in COPD and the potential pulmonary protective mechanisms of TXL in COPD complicated with AS. COPD complicated with AS was induced in mice by cigarette smoke (CS) exposure and high-fat diet (HFD) feeding. The mice were treated with atorvastatin (ATO), TXL or combination therapy (ATO+TXL) for 20 weeks. Pulmonary function, lung pathology, serum lipid levels, atherosclerotic plaque area and indicators of barrier function, oxidative stress and ferroptosis in lung tissue were evaluated. In vitro, human pulmonary microvascular endothelial cells (HPMECs) were pretreated with TXL for 4 h and then incubated with cigarette smoke extract (CSE) and homocysteine (Hcy) for 36 h to induce barrier dysfunction. Then the indicators of barrier function, oxidative stress and ferroptosis were measured. The results demonstrate that CS aggravated dyslipidaemia, atherosclerotic plaque formation, pulmonary function decline, pathological injury, barrier dysfunction, oxidative stress and ferroptosis in the HFD-fed mice. However, these abnormalities were partially reversed by ATO and TXL. Similar results were observed in vitro. In conclusion, pulmonary microvascular barrier dysfunction plays an important role by which COPD affects the progression of AS, and ferroptosis may be involved. Moreover, TXL delays the progression of AS and reduces cardiovascular events by protecting the pulmonary microvascular barrier and inhibiting ferroptosis.

Si-Miao-Yong-An Decoction Maintains the Cardiac Function and Protects Cardiomyocytes from Myocardial Ischemia and Reperfusion Injury.

OBJECTIVE: The aim of this study was to determine whether Si-Miao-Yong-An decoction (SMYAD) could protect cardiomyocytes from ischemia/reperfusion (I/R) injury and its underlying mechanisms. METHODS: C57BL/6 mice were used to establish a model of myocardial infarction by I/R injury and treated by SMYAD for 4 weeks. Then, the cardiac functions of mice were evaluated by cardiac magnetic resonance (CMR). Histopathological analysis for the heart remodeling was detected by H&E and Masson staining. The protein expression of collagen I, MMP9, and TNFα was detected by western blot in the heart tissues. H9C2 cells were used to establish the hypoxia/reoxygenation (H/R) model and SMYAD intervention. MTT assays detected the cell viability of myocardial cells. The expression level of IL-1ß was evaluated by ELISA. The expression levels of LC3B-II/LC3B-I, p-mTOR, mTOR, NLRP3, procaspase 1, and cleaved-caspase 1 in H9C2 cells were evaluated by Western blot. RESULTS: SMYAD improved cardiac functions such as ventricular volume and ejection fraction of the rats with ischemia/reperfusion injury. Morphological assay indicated that SMYAD reduced the scar size and inhibited fibrosis formation. It was found that SMYAD could regulate collagen I, MMP9, and TNFα protein expression levels in the heart tissues. SMYAD improved the survival rate of H9C2 cardiomyocytes in the H/R injury model. SMYAD elevated the rate of LC3B-II/LC3B-I protein expression, decreased the rate of p-mTOR/mTOR protein expression, and reduced expressions of caspase 1, NLRP3, and IL-1ß in H/R cardiomyocytes. CONCLUSION: SMYAD exerted protective effects on ischemia/reperfusion injury in myocardial cells by activating autophagy and inhibiting pyroptosis. This might be the reason why SMYAD protected myocardial tissue and improved cardiac function in mice with ischemia/reperfusion.

Acupuncture at PC6 prevents cardiac hypertrophy in isoproterenol-treated mice.

OBJECTIVES: To investigate the effect of acupuncture at PC6 on cardiac hypertrophy in isoproterenol (ISO)-treated mice. METHODS: 48 male C57BL/6 mice underwent subcutaneous injection of ISO for 14 days and were randomly divided into four groups (n=12 each) that remained untreated (ISO group), received verum manual acupuncture (MA) treatment at PC6 (ISO+MA(PC6) group), sham MA at location on the tail not corresponding to any traditional acupuncture point (ISO+MA(tail) group), or propranolol (ISO+PR group). An additional 12 mice were given an injection of phosphate-buffered saline (PBS) and formed a healthy control (Normal) group. After performing echocardiography and measuring the ratio of heart weight (HW)/tibia length (TL) at 14 days, all mice were euthanased. Morphological examination was performed following haematoxylin and eosin and Masson's staining of heart tissues. Ultrastructural changes were observed by electron microscopy. Cardiac protein expression of atrial natriuretic peptide (ANP) and tumour necrosis factor α (TNFα) were measured by immunohistochemical (IHC) staining and Western blotting. RESULTS: Compared with the untreated model group, acupuncture at PC6 lowered the heart rate, reduced the ratio of HW/TL, improved the left ventricular (LV) anterior wall thickness (LVAWd), LV end-diastolic anterior wall thickness (LVAWs), LV end-systolic posterior wall thickness (LVPWd), LV end-diastolic posterior wall thickness (LVPWs), and fractional shortening (FS) as observed by echocardiography (ISO+MA(PC6) vs. ISO groups: P<0.05). Moreover, evidence from morphological studies demonstrated that acupuncture at PC6 inhibited myocardial hypertrophy and collagen deposition, and normalised the ultrastructural changes. In addition, ANP and TNFα expression were attenuated in the verum acupuncture group compared with the untreated model group (ISO+MA(PC6) vs. ISO groups: P<0.05). CONCLUSIONS: The results demonstrated that acupuncture at PC6 attenuates sympathetic overactivity. Additionally, it may improve cardiac performance by reversing adverse cardiac remodelling. Acupuncture has potential as a treatment for sympathetic hypertension.

Animal Models and "Omics" Technologies for Identification of Novel Biomarkers and Drug Targets to Prevent Heart Failure.

It is now accepted that heart failure (HF) is a complex multifunctional disease rather than simply a hemodynamic dysfunction. Despite its complexity, stressed cardiomyocytes often follow conserved patterns of structural remodelling in order to adapt, survive, and regenerate. When cardiac adaptations cannot cope with mechanical, ischemic, and metabolic loads efficiently or become chronically activated, as, for example, after infection, then the ongoing structural remodelling and dedifferentiation often lead to compromised pump function and patient death. It is, therefore, of major importance to understand key events in the progression from a compensatory left ventricular (LV) systolic dysfunction to a decompensatory LV systolic dysfunction and HF. To achieve this, various animal models in combination with an "omics" toolbox can be used. These approaches will ultimately lead to the identification of an arsenal of biomarkers and therapeutic targets which have the potential to shape the medicine of the future.

Spiroalkaloids from Shensong Yangxin capsule.

Four spiroalkaloids, including a new compound shensongine A (1), were isolated from the anti-arrhythmic TCM formula Shensong Yangxin capsule. Their structures were determined on the basis of spectroscopic analysis. Compounds 1 and 3 displayed cardiovascular activities by shortened APD in rat myocardial cells. These compounds were possibly generated from precursors in different composed herbal medicines during the processing of the TCM formula.

A combination of genistein and magnesium enhances the vasodilatory effect via an eNOS pathway and BK(Ca) current amplification.

The phytoestrogen genistein (GST) and magnesium have been independently shown to regulate vascular tone; however, their individual vasodilatory effects are limited. The aim of this study was to examine the combined effects of GST plus magnesium on vascular tone in mesenteric arteries. The effects of pretreatment with GST (0-200 µmol/L), MgCl2 (0-4.8 mmol/L) and GST plus MgCl2 on 10 µmol/L phenylephrine (PE) precontracted mesenteric arteries in rats were assessed by measuring isometric force. BK(Ca) currents were detected by the patch clamp method. GST caused concentration- and partial endothelium-dependent relaxation. Magnesium resulted in dual adjustment of vascular tone. Magnesium-free solution eliminated the vasodilatation of GST in both endothelium-intact and denuded rings. GST (50 µmol/L) plus magnesium (4.8 mmol/L) caused stronger relaxation in both endothelium-intact and denuded rings. Pretreatment with the nitric oxide synthase (NOS) inhibitor L-N-nitroarginine methyl ester (L-NAME, 100 µmol/L) significantly inhibited the effects of GST, high magnesium, and the combination of GST and magnesium. BK(Ca) currents were amplified to a greater extent when GST (50 µmol/L) was combined with 4.8 versus 1.2 mmol/L Mg(2+). Our data suggest that GST plus magnesium provides enhanced vasodilatory effects in rat mesenteric arteries compared with that observed when either is used separately, which was related to an eNOS pathway and BK(Ca) current amplification.