OBJECTIVE: To Investigate the effects of lithocholic acid (LCA) on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). METHODS: Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group (undergoing bilateral ovariectomy) and a control group (only removing the same volume of adipose tissue around the ovaries), with 6 mice in each group. The body mass was measured every week after operation. After 4 weeks post-surgery, the weight of mouse uterus was measured, femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass. Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area. ELISA was used to detect the expression of bone turnover markers in the serum. Liver samples were subjected to real-time fluorescence quantitative PCR (RT-qPCR) to detect the expression of key genes related to bile acid metabolism, including cyp7a1, cyp7b1, cyp8b1, and cyp27a1. BMSCs were isolated by centrifugation from 2 C57BL/6J female mice (10-week-old). The third-generation cells were exposed to 0, 1, 10, and 100 μmol/L LCA, following which cell viability was evaluated using the cell counting kit 8 assay. Subsequently, alkaline phosphatase (ALP) staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction. RT-qPCR was employed to analyze the expressions of osteogenic-related genes, namely ALP, Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), as well as adipogenic-related genes including Adiponectin (Adipoq), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor γ (PPARγ). RESULTS: Compared with the control group, the body mass of the mice in the experimental group increased, the uterus atrophied, the bone mass decreased, the bone marrow fat expanded, and the bone metabolism showed a high bone turnover state. RT-qPCR showed that the expressions of cyp7a1, cyp8b1, and cyp27a1, which were related to the key enzymes of bile acid metabolism in the liver, decreased significantly ( P<0.05), while the expression of cyp7b1 had no significant difference ( P>0.05). Intervention with LCA at concentrations of 1, 10, and 100 μmol/L did not demonstrate any apparent toxic effects on BMSCs. Furthermore, LCA inhibited the expressions of osteogenic-related genes (ALP, Runx2, and OCN) in a dose-dependent manner, resulting in a reduction in ALP staining positive area. Concurrently, LCA promoted the expressions of adipogenic-related genes (Adipoq, FABP4, and PPARγ), and an increase in oil red O staining positive area. CONCLUSION After menopause, the metabolism of bile acids is altered, and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs, thereby affecting bone remodelling.
Female ; Mice ; Animals ; Core Binding Factor Alpha 1 Subunit/pharmacology* ; PPAR gamma/metabolism* ; Steroid 12-alpha-Hydroxylase/metabolism* ; Mice, Inbred C57BL ; Cell Differentiation ; Osteogenesis ; Mesenchymal Stem Cells ; Bile Acids and Salts/pharmacology* ; Bone Marrow Cells ; Cells, Cultured ; Azo Compounds

Humans ; PPAR gamma/metabolism* ; Multiple Sclerosis ; Transcription Factors/metabolism* ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha

OBJECTIVE: To observe the effect of Tongdu Tiaoshen (promoting the circulation of the governor vessel and regulating the spirit) electroacupuncture (EA) pretreatment on pyroptosis mediated by peroxisome proliferators-activated receptor γ (PPARγ) of the cerebral cortex in rats with cerebral ischemia reperfusion injury (CIRI) and explore the potential mechanism of EA for the prevention and treatment of CIRI. METHODS: A total of 110 clean-grade male SD rats were randomly divided into a sham-operation group, a model group, an EA group, an EA + inhibitor group and an agonist group, 22 rats in each group. In the EA group, before modeling, EA was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14), with disperse-dense wave, 2 Hz/5 Hz in frequency, 1 to 2 mA in intensity, lasting 20 min; once a day, consecutively for 7 days. On the base of the intervention as the EA group, on the day 7, the intraperitoneal injection with the PPARγ inhibitor, GW9662 (10 mg/kg) was delivered in the EA + inhibitor group. In the agonist group, on the day 7, the PPARγ agonist, pioglitazone hydrochloride (10 mg/kg) was injected intraperitoneally. At the end of intervention, except the sham-operation group, the modified thread embolization method was adopted to establish the right CIRI model in the rats of the other groups. Using the score of the modified neurological severity score (mNSS), the neurological defect condition of rats was evaluated. TTC staining was adopted to detect the relative cerebral infarction volume of rat, TUNEL staining was used to detect apoptosis of cerebral cortical nerve cells and the transmission electron microscope was used to observe pyroptosis of cerebral cortical neural cells. The positive expression of PPARγ and nucleotide-binding to oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex was detected with the immunofluorescence staining. The protein expression of PPARγ, NLRP3, cysteinyl aspartate specific protease-1 (caspase-1), gasdermin D (GSDMD) and GSDMD-N terminal (GSDMD-N) in the cerebral cortex was detected with Western blot. Using the quantitative real-time fluorescence-PCR, the mRNA expression of PPARγ, NLRP3, caspase-1 and GSDMD of the cerebral cortex was detected. The contents of interleukin (IL)-1β and IL-18 in the cerebral cortex of rats were determined by ELISA. RESULTS: Compared with the sham-operation group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.01), pyroptosis was severe, the protein and mRNA expression levels of PPARγ, NLRP3, caspase-1 and GSDMD were elevated (P<0.01); and the protein expression of GSDMD-N and contents of IL-1β and IL-18 were increased (P<0.01) in the model group. When compared with the model group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1β and IL-18 were lower (P<0.01) in the EA group and the agonist group; while, in the EA + inhibitor group, the protein expression of PPARγ was increased (P<0.01), the protein and mRNA expression levels of NLRP3 and GSDMD were decreased (P<0.01, P<0.05), the mRNA expression of caspase-1 was reduced (P<0.01); and the contents of IL-1β and IL-18 were lower (P<0.01). When compared with the EA + inhibitor group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.05, P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1β and IL-18 were declined (P<0.01) in the EA group. Compared with the agonist group, in the EA group, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.05, P<0.01), the mRNA expression of PPARγ was decreased (P<0.01) and the protein expression of GSDMD-N was elevated (P<0.05); and the contents of IL-1β and IL-18 were higher (P<0.01). CONCLUSION Tongdu Tiaoshen EA pretreatment can attenuate the neurological impairment in the rats with CIRI, and the underlying mechanism is related to the up-regulation of PPARγ inducing the inhibition of NLRP3 in the cerebral cortex of rats so that pyroptosis is affected.
Male ; Animals ; Rats ; Rats, Sprague-Dawley ; PPAR gamma/genetics* ; Pyroptosis ; Interleukin-18 ; Electroacupuncture ; NLR Family, Pyrin Domain-Containing 3 Protein ; Cerebral Cortex ; Cerebral Infarction/therapy* ; Caspases ; RNA, Messenger

Hepatic lipid deposition is one of the basic manifestations of obesity, and nowadays pharmacological treatment is the most important tool. Punicalagin(PU), a polyphenol derived from pomegranate peel, is a potential anti-obesity substance. In this study, 60 C57BL/6J mice were randomly divided into a normal group and a model group. After establishing a model of simple obesity with a high-fat diet for 12 weeks, the successfully established rat models of obesity were then regrouped into a model group, an orlistat group, a PU low-dose group, a PU medium-dose group, and a PU high-dose group. The normal group was kept on routine diet and other groups continued to feed the high-fat diet. The body weight and food intake were measured and recorded weekly. After 8 weeks, the levels of the four lipids in the serum of each group of mice were determined by an automatic biochemical instrument. Oral glucose tole-rance and intraperitoneal insulin sensitivity were tested. Hemoxylin-eosin(HE) staining was applied to observe the hepatic and adipose tissues. The mRNA expression levels of peroxisome proliferators-activated receptor γ(PPARγ) and C/EBPα were determined by real-time quantitative polymerase chain reaction(Q-PCR), and the mRNA and protein expression levels of adenosine 5'-monophosphate-activated protein kinase(AMPK), anterior cingulate cortex(ACC), and carnitine palmitoyltransferase 1A(CPT1A) were determined by Western blot. Finally, the body mass, Lee's index, serum total glyceride(TG), serum total cholesterol(TC), and low-density lipoprotein cholesterol(LDL-C) levels were significantly higher and high-density lipoprotein cholesterol(HDL-C) levels were significantly lower in the model group as compared with the normal group. The fat deposition in the liver was significantly increased. The mRNA expression levels of hepatic PPARγ and C/EBPα and the protein expression level of ACC were increased, while the mRNA and protein expression levels of CPT-1α(CPT1A) and AMPK were decreased. After PU treatment, the above indexes of obese mice were reversed. In conclusion, PU can decrease the body weight of obese mice and control their food intake. It also plays a role in the regulation of lipid metabolism and glycometabolism metabolism, which can significantly improve hepatic fat deposition. Mechanistically, PU may regulate liver lipid deposition in obese mice by down-regulating lipid synthesis and up-regulating lipolysis through activation of the AMPK/ACC pathway.
Rats ; Mice ; Animals ; Mice, Obese ; AMP-Activated Protein Kinases/metabolism* ; PPAR gamma/metabolism* ; Mice, Inbred C57BL ; Liver/metabolism* ; Obesity/genetics* ; Body Weight ; Lipid Metabolism ; Diet, High-Fat/adverse effects* ; Lipids ; Cholesterol

OBJECTIVE: To investigate the effect of adipocytes in the bone marrow microenvironment of patients with multiple myeloma (MM) on the pathogenesis of MM. METHODS: Bone marrow adipocytes (BMA) in bone marrow smears of health donors (HD) and newly diagnosed MM (ND-MM) patients were evaluated with oil red O staining. The mesenchymal stem cells (MSC) from HD and ND-MM patients were isolated, and in vitro co-culture assay was used to explore the effects of MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells. The expression of adipogenic/osteogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4, FASN and ALP both in MSC and MSC-derived adipocytes was determined with real-time quantitative PCR. The Western blot was employed to detect the expression levels of IL-6, IL-10, SDF-1α, TNF-α and IGF-1 in the supernatant with or without PPAR-γ inhibitor. RESULTS: The results of oil red O staining of bone marrow smears showed that BMA increased significantly in patients of ND-MM compared with the normal control group, and the BMA content was related to the disease status. The content of BMA decreased in the patients with effective chemotherapy. MM cells up-regulated the expression of MSC adipogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4 and FASN, but the expression of osteogenic differentiation-related gene ALP was significantly down-regulated. This means that the direct consequence of the interaction between MM cells and MSC in the bone marrow microenvironment is to promote the differentiation of MSC into adipocytes at the expense of osteoblasts, and the cytokines detected in supernatant changed. PPAR-γ inhibitor G3335 could partially reverse the release of cytokines by BMA. Those results confirmed that BMA regulated the release of cytokines via PPAR-γ signal, and PPAR-γ inhibitor G3335 could distort PPAR-γ mediated BMA maturation and cytokines release. The increased BMA and related cytokines effectively promoted the proliferation, migration and drug resistance of MM cells. CONCLUSION The BMA and its associated cytokines are the promoting factors in the survival, proliferation and migration of MM cells. BMA can protect MM cells from drug-induced apoptosis and plays an important role in MM treatment failure and disease progression.
Humans ; Osteogenesis/genetics* ; Bone Marrow/metabolism* ; Multiple Myeloma/metabolism* ; Drug Resistance, Neoplasm ; Peroxisome Proliferator-Activated Receptors/pharmacology* ; Cell Differentiation ; Adipogenesis ; Cytokines/metabolism* ; Adipocytes/metabolism* ; Bone Marrow Cells/metabolism* ; Cells, Cultured ; PPAR gamma/pharmacology* ; Tumor Microenvironment

Melatonin receptor 1B (MT2, encoded by the MTNR1B gene), a high-affinity receptor for melatonin, is associated with glucose homeostasis including glucose uptake and transport. The rs10830963 variant in the MTNR1B gene is linked to glucose metabolism disorders including gestational diabetes mellitus (GDM); however, the relationship between MT2-mediated melatonin signaling and a high birth weight of GDM infants from maternal glucose abnormality remains poorly understood. This article aims to investigate the relationship between rs10830963 variants and GDM development, as well as the effects of MT2 receptor on glucose uptake and transport in trophoblasts. TaqMan-MGB (minor groove binder) probe quantitative real-time polymerase chain reaction (qPCR) assays were used for rs10930963 genotyping. MT2 expression in the placenta of GDM and normal pregnant women was detected by immunofluorescence, western blot, and qPCR. The relationship between MT2 and glucose transporters (GLUTs) or peroxisome proliferator-activated receptor γ (PPARγ) was established by western blot, and glucose consumption of trophoblasts was measured by a glucose assay kit. The results showed that the genotype and allele frequencies of rs10830963 were significantly different between GDM and normal pregnant women (P<0.05). The fasting, 1-h and 2-h plasma glucose levels of G-allele carriers were significantly higher than those of C-allele carriers (P<0.05). Besides, the protein and messenger RNA (mRNA) expression of MT2 in the placenta of GDM was significantly higher than that of normal pregnant women (P<0.05). Melatonin could stimulate glucose uptake and GLUT4 and PPARγ protein expression in trophoblasts, which could be attenuated by MT2 receptor knockdown. In conclusion, the rs10830963 variant was associated with an increased risk of GDM. The MT2 receptor is essential for melatonin to raise glucose uptake and transport, which may be mediated by PPARγ.
Female ; Humans ; Pregnancy ; Blood Glucose/metabolism* ; Diabetes, Gestational/metabolism* ; Glucose/metabolism* ; Melatonin/metabolism* ; Polymorphism, Genetic ; PPAR gamma ; Receptor, Melatonin, MT2/genetics*

OBJECTIVE: To establish a simple, low-cost and time-saving method for primary culture of mature white adipocytes from mice. METHODS: Mature white adipocytes were isolated from the epididymis and perirenal area of mice for primary culture using a modified mature adipocyte culture method or the ceiling culture method. The morphology of the cultured mature adipocytes was observed using Oil Red O staining, and the cell viability was assessed with CCK8 method. The expression of PPARγ protein in the cells was detected with Western blotting, and the mRNA expressions of CD36, FAS, CPT1A and FABP4 were detected using RT-qPCR. RESULTS: Oil Red O staining showed a good and uniform morphology of the adipocytes in primary culture using the modified culture method, while the cells cultured using the ceiling culture method exhibited obvious morphological changes. CCK8 assay showed no significant difference in cell viability between freshly isolated mature white adipocytes and the cells obtained with the modified culture method. Western blotting showed that the freshly isolated adipocytes and the cells cultured for 72 h did not differ significantly in the expression levels of PPARγ protein (P=0.759), which was significantly lowered in response to treatment with GW9662 (P < 0.001). GW9662 treatment of the cells upregulated mRNA expressions of CD36 (P < 0.001) and CPT1A (P=0.003) and down-regulated those of FAS (P=0.001) and FABP4 (P < 0.001). CONCLUSION We established a convenient and time-saving method for primary culture mature white adipocytes from mice to facilitate further functional studies of mature adipocytes.
Male ; Mice ; Animals ; Adipocytes, White/metabolism* ; PPAR gamma/metabolism* ; RNA, Messenger ; Cell Differentiation ; 3T3-L1 Cells

Xenogenic organ transplantation has been considered the most promising strategy in providing possible substitutes with the physiological function of the failing organs as well as solving the problem of insufficient donor sources. However, the xenograft, suffered from immune rejection and ischemia-reperfusion injury (IRI), causes massive reactive oxygen species (ROS) expression and the subsequent cell apoptosis, leading to the xenograft failure. Our previous study found a positive role of PPAR-γ in anti-inflammation through its immunomodulation effects, which inspires us to apply PPAR-γ agonist rosiglitazone (RSG) to address survival issue of xenograft with the potential to eliminate the excessive ROS. In this study, xenogenic bioroot was constructed by wrapping the dental follicle cells (DFC) with porcine extracellular matrix (pECM). The hydrogen peroxide (H₂O₂)-induced DFC was pretreated with RSG to observe its protection on the damaged biological function. Immunoflourescence staining and transmission electron microscope were used to detect the intracellular ROS level. SD rat orthotopic transplantation model and superoxide dismutase 1 (SOD1) knockout mice subcutaneous transplantation model were applied to explore the regenerative outcome of the xenograft. It showed that RSG pretreatment significantly reduced the adverse effects of H₂O₂ on DFC with decreased intracellular ROS expression and alleviated mitochondrial damage. In vivo results confirmed RSG administration substantially enhanced the host's antioxidant capacity with reduced osteoclasts formation and increased periodontal ligament-like tissue regeneration efficiency, maximumly maintaining the xenograft function. We considered that RSG preconditioning could preserve the biological properties of the transplanted stem cells under oxidative stress (OS) microenvironment and promote organ regeneration by attenuating the inflammatory reaction and OS injury.
Mice ; Humans ; Rats ; Animals ; Swine ; PPAR gamma/pharmacology* ; Reactive Oxygen Species/pharmacology* ; Heterografts ; Hydrogen Peroxide/pharmacology* ; Rats, Sprague-Dawley ; Rosiglitazone/pharmacology* ; Oxidative Stress

Diabetes mellitus is a chronic disease, typified by hyperglycemia resulting from failures in complex multifactorial metabolic functions, that requires life-long medication. Prolonged uncontrolled hyperglycemia leads to micro- and macro-vascular complications. Although antidiabetic drugs are prescribed as the first-line treatment, many of them lose efficacy over time or have severe side effects. There is a lack of in-depth study on the patents filed concerning the use of natural compounds to manage diabetes. Thus, this patent analysis provides a comprehensive report on the antidiabetic therapeutic activity of 6 phytocompounds when taken alone or in combinations. Four patent databases were searched, and 17,649 patents filed between 2001 and 2021 were retrieved. Of these, 139 patents for antidiabetic therapeutic aids that included berberine, curcumin, gingerol, gymnemic acid, gymnemagenin and mangiferin were analyzed. The results showed that these compounds alone or in combinations, targeting acetyl-coenzyme A carboxylase 2, serine/threonine protein kinase, α-amylase, α-glucosidase, lipooxygenase, phosphorylase, peroxisome proliferator-activated receptor-γ (PPARγ), protein tyrosine phosphatase 1B, PPARγ co-activator-1α, phosphoinositide 3-kinase and protein phosphatase 1 regulatory subunit 3C, could regulate glucose metabolism which are validated by pharmacological rationale. Synergism, or combination therapy, including different phytocompounds and plant extracts, has been studied extensively and found effective, whereas the efficacy of commercial drugs in combination with phytocompounds has not been studied in detail. Curcumin, gymnemic acid and mangiferin were found to be effective against diabetes-related complications. Please cite this article as: DasNandy A, Virge R, Hegde HV, Chattopadhyay D. A review of patent literature on the regulation of glucose metabolism by six phytocompounds in the management of diabetes mellitus and its complications. J Integr Med. 2023; 21(3): 226-235.
Humans ; PPAR gamma/metabolism* ; Curcumin/therapeutic use* ; Phosphatidylinositol 3-Kinases ; Diabetes Mellitus/drug therapy* ; Hypoglycemic Agents/pharmacology* ; Hyperglycemia/drug therapy* ; Glucose

This paper aimed to investigate the effect of total flavonoids of buckwheat flower and leaf on myocardial cell apoptosis and Wnt/β-catenin/peroxisome proliferator-activated receptor γ(PPARγ) pathway in arrhythmic rats. SD rats were randomly divided into a control group, a model group, a low-dose(20 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a medium-dose(40 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a high-dose(80 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a propranolol hydrochloride(2 mg·kg~(-1)) group, with 12 rats in each group. Except the control group, rats in other groups were prepared as models of arrhythmia by sublingual injection of 1 mL·kg~(-1) of 0.002% aconitine. After grouping and intervention with drugs, the arrhythmia, myocardial cells apoptosis, myocardial tissue glutathione peroxidase(GSH-Px), catalase(CAT), malondialdehyde(MDA), serum interleukin-6(IL-6), prostaglandin E2(PGE2) levels, myocardial tissue apoptosis, and Wnt/β-catenin/PPARγ pathway-related protein expression of rats in each group were measured. As compared with the control group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA levels in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels increased significantly in the model group, whereas the GSH-Px and CAT levels, and Bcl-2 and PPARγ protein expression levels in myocardial tissues reduced significantly. As compared with the model group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA leve in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels reduced in the drug intervention groups, whereas the GSH-Px and CAT levels and Bcl-2 and PPARγ protein expression levels in myocardial tissues increased. The groups of total flavonoids of buckwheat flower and leaf were in a dose-dependent manner. There was no significant difference in the levels of each index in rats between the propranolol hydrochloride group and the high-dose group of total flavonoids of buckwheat flower and leaf. The total flavonoids of buckwheat flower and leaf inhibit the activation of Wnt/β-catenin pathway, up-regulate the expression of PPARγ, reduce oxidative stress and inflammatory damage in myocardial tissues of arrhythmic rats, reduce myocardial cell apoptosis, and improve the symptoms of arrhythmia in rats.
Rats ; Animals ; PPAR gamma/metabolism* ; Fagopyrum/genetics* ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; beta Catenin/metabolism* ; Interleukin-6 ; Flavonoids/pharmacology* ; Propranolol/pharmacology* ; Ventricular Fibrillation ; Dinoprostone ; Wnt Signaling Pathway ; Plant Leaves/metabolism* ; Flowers/metabolism* ; Apoptosis ; Cardiac Complexes, Premature