Muscle-bone cross-talk through the FNIP1-TFEB-IGF2 axis is associated with bone metabolism in human and mouse.

Clinical evidence indicates a close association between muscle dysfunction and bone loss; however, the underlying mechanisms remain unclear. Here, we report that muscle dysfunction-related bone loss in humans with limb-girdle muscular dystrophy is associated with decreased expression of folliculin-interacting protein 1 (FNIP1) in muscle tissue. Supporting this finding, murine gain- and loss-of-function genetic models demonstrated that muscle-specific ablation of FNIP1 caused decreased bone mass, increased osteoclastic activity, and mechanical impairment that could be rescued by myofiber-specific expression of FNIP1. Myofiber-specific FNIP1 deficiency stimulated expression of nuclear translocation of transcription factor EB, thereby activating transcription of insulin-like growth factor 2 (Igf2) at a conserved promoter-binding site and subsequent IGF2 secretion. Muscle-derived IGF2 stimulated osteoclastogenesis through IGF2 receptor signaling. AAV9-mediated overexpression of IGF2 was sufficient to decrease bone volume and impair bone mechanical properties in mice. Further, we found that serum IGF2 concentration was negatively correlated with bone health in humans in the context of osteoporosis. Our findings elucidate a muscle-bone cross-talk mechanism bridging the gap between muscle dysfunction and bone loss. This cross-talk represents a potential target to treat musculoskeletal diseases and osteoporosis.

TKT-PARP1 axis induces radioresistance by promoting DNA double-strand break repair in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) stands as the fifth most prevalent malignant tumor on a global scale and presents as the second leading cause of cancer-related mortality. DNA damage-based radiotherapy (RT) plays a pivotal role in the treatment of HCC. Nevertheless, radioresistance remains a primary factor contributing to the failure of radiation therapy in HCC patients. In this study, we investigated the functional role of transketolase (TKT) in the repair of DNA double-strand breaks (DSBs) in HCC. Our research unveiled that TKT is involved in DSB repair, and its depletion significantly reduces both non-homologous end joining (NHEJ) and homologous recombination (HR)-mediated DSB repair. Mechanistically, TKT interacts with PARP1 in a DNA damage-dependent manner. Furthermore, TKT undergoes PARylation by PARP1, resulting in the inhibition of its enzymatic activity, and TKT can enhance the auto-PARylation of PARP1 in response to DSBs in HCC. The depletion of TKT effectively mitigates the radioresistance of HCC, both in vitro and in mouse xenograft models. Moreover, high TKT expression confers resistance of RT in clinical HCC patients, establishing TKT as a marker for assessing the response of HCC patients who received cancer RT. In summary, our findings reveal a novel mechanism by which TKT contributes to the radioresistance of HCC. Overall, we identify the TKT-PARP1 axis as a promising potential therapeutic target for improving RT outcomes in HCC.

FNIP1 abrogation promotes functional revascularization of ischemic skeletal muscle by driving macrophage recruitment.

Ischaemia of the heart and limbs attributable to compromised blood supply is a major cause of mortality and morbidity. The mechanisms of functional angiogenesis remain poorly understood, however. Here we show that FNIP1 plays a critical role in controlling skeletal muscle functional angiogenesis, a process pivotal for muscle revascularization during ischemia. Muscle FNIP1 expression is down-regulated by exercise. Genetic overexpression of FNIP1 in myofiber causes limited angiogenesis in mice, whereas its myofiber-specific ablation markedly promotes the formation of functional blood vessels. Interestingly, the increased muscle angiogenesis is independent of AMPK but due to enhanced macrophage recruitment in FNIP1-depleted muscles. Mechanistically, myofiber FNIP1 deficiency induces PGC-1α to activate chemokine gene transcription, thereby driving macrophage recruitment and muscle angiogenesis program. Furthermore, in a mouse hindlimb ischemia model of peripheral artery disease, the loss of myofiber FNIP1 significantly improved the recovery of blood flow. Thus, these results reveal a pivotal role of FNIP1 as a negative regulator of functional angiogenesis in muscle, offering insight into potential therapeutic strategies for ischemic diseases.

iTRAQ Quantitative Proteomic Analysis of Different Expressed Proteins and Signal Pathways in Bakuchiol-Induced Hepatotoxicity.

Bakuchiol (BAK) is an abundant natural compound. BAK has been reported to have several biological activities such as anticancer, antiaging, anti-inflammatory, and prevention of bone loss. However, it causes hepatotoxicity, the mechanism of which is not known. In this study, we explored the mechanism of BAK hepatotoxicity by treating rats with 52.5 mg/kg and 262.5 mg/kg of BAK, administered continuously for 6 weeks. We examined the liver pathology and biochemical composition of bile to determine toxicity. Mechanisms of BAK hepatotoxicity were analyzed based on relative and absolute quantification (iTRAQ) protein equivalent signatures and validated in vitro using LO2 cells. iTRAQ analysis revealed 281 differentially expressed proteins (DEPs) in liver tissue of the BAK-treated group, of which 215 were upregulated, and 66 were downregulated. GO and KEGG enrichment analysis revealed that bile secretion, lipid metabolism, and cytochrome P450 signaling pathways were enriched in DEPs. Among them, peroxisome proliferator-activated receptor α (PPARα), farnesoid X receptor (FXR), and cholesterol 7α-hydroxylase (CYP7a1) were closely associated with the development and progression of BAK-induced hepatic metabolic dysfunction and abnormal bile metabolism. This study shows that BAK can induce hepatotoxicity through multiple signaling pathways.

FNIP1 regulates adipocyte browning and systemic glucose homeostasis in mice by shaping intracellular calcium dynamics.

Metabolically beneficial beige adipocytes offer tremendous potential to combat metabolic diseases. The folliculin interacting protein 1 (FNIP1) is implicated in controlling cellular metabolism via AMPK and mTORC1. However, whether and how FNIP1 regulates adipocyte browning is unclear. Here, we demonstrate that FNIP1 plays a critical role in controlling adipocyte browning and systemic glucose homeostasis. Adipocyte-specific ablation of FNIP1 promotes a broad thermogenic remodeling of adipocytes, including increased UCP1 levels, high mitochondrial content, and augmented capacity for mitochondrial respiration. Mechanistically, FNIP1 binds to and promotes the activity of SERCA, a main Ca2+ pump responsible for cytosolic Ca2+ removal. Loss of FNIP1 resulted in enhanced intracellular Ca2+ signals and consequential activation of Ca2+-dependent thermogenic program in adipocytes. Furthermore, mice lacking adipocyte FNIP1 were protected against high-fat diet-induced insulin resistance and liver steatosis. Thus, these findings reveal a pivotal role of FNIP1 as a negative regulator of beige adipocyte thermogenesis and unravel an intriguing functional link between intracellular Ca2+ dynamics and adipocyte browning.

A new therapeutic effect of fenofibrate in Duchenne muscular dystrophy: The promotion of myostatin degradation.

BACKGROUND AND PURPOSE: Duchenne muscular dystrophy (DMD) is a degenerative muscle disease with no effective drug treatment. This study investigated the positive effects of fenofibrate on dystrophic muscles. EXPERIMENTAL APPROACH: Myostatin expression in serum and muscle tissue from patients with Duchenne muscular dystrophy and mdx mice were tested. Primary myoblasts isolated from mdx mice were challenged with an inflammatory stimulus and treated with fenofibrate. In animal experiments, 6-week-old male mdx mice were treated with fenofibrate (100 mg kg-1 ) administered orally once per day for 6 weeks. Effects of fenofibrate were evaluated by tests of muscle function plus histology and biochemical analyses of serum. Expression of myostatin, MuRF1, and atrogin-1 in skeletal muscle was evaluated by western blotting and real-time PCR. Total and oxidative myosin heavy chain (MHC) were assessed via immunofluorescence. KEY RESULTS: Expression of myostatin protein was increased in dystrophic muscle of patients with Duchenne muscular dystrophy and mdx mice. Fenofibrate enhanced myofibre differentiation by down-regulating the expression of myostatin protein but not mRNA in primary myoblasts of mdx mice. Fenofibrate significantly improved muscle function while ameliorating muscle damage in mdx mice. These benefits were accompanied by an anti-inflammatory effect. Fenofibrate treatment returned myofibre function by inhibiting the expressions of myostatin, MuRF1, and atrogin-1 protein in the gastrocnemius muscle and diaphragm, while leaving the mRNA level of myostatin unaffected. CONCLUSIONS AND IMPLICATIONS: Fenofibrate substantially slows muscle dystrophy by promoting the degradation of myostatin protein, which may indicate a new therapeutic focus for patients with Duchenne muscular dystrophy.

IRE1α regulates skeletal muscle regeneration through Myostatin mRNA decay.

Skeletal muscle can undergo a regenerative process from injury or disease to preserve muscle mass and function, which is critically influenced by cellular stress responses. Inositol-requiring enzyme 1 (IRE1) is an ancient endoplasmic reticulum (ER) stress sensor and mediates a key branch of the unfolded protein response (UPR). In mammals, IRE1α is implicated in the homeostatic control of stress responses during tissue injury and regeneration. Here, we show that IRE1α serves as a myogenic regulator in skeletal muscle regeneration in response to injury and muscular dystrophy. We found in mice that IRE1α was activated during injury-induced muscle regeneration, and muscle-specific IRE1α ablation resulted in impaired regeneration upon cardiotoxin-induced injury. Gain- and loss-of-function studies in myocytes demonstrated that IRE1αacts to sustain both differentiation in myoblasts and hypertrophy in myotubes through regulated IRE1-dependent decay (RIDD) of mRNA encoding Myostatin, a key negative regulator of muscle repair and growth. Furthermore, in the mouse model of Duchenne muscular dystrophy (DMD), loss of muscle IRE1α resulted in augmented Myostatin signaling and exacerbated the dystrophic phenotypes. Thus, these results reveal a pivotal role for the RIDD output of IRE1α in muscle regeneration, offering new insight into potential therapeutic strategies for muscle loss diseases.

AMPK-dependent and -independent coordination of mitochondrial function and muscle fiber type by FNIP1.

Mitochondria are essential for maintaining skeletal muscle metabolic homeostasis during adaptive response to a myriad of physiologic or pathophysiological stresses. The mechanisms by which mitochondrial function and contractile fiber type are concordantly regulated to ensure muscle function remain poorly understood. Evidence is emerging that the Folliculin interacting protein 1 (Fnip1) is involved in skeletal muscle fiber type specification, function, and disease. In this study, Fnip1 was specifically expressed in skeletal muscle in Fnip1-transgenic (Fnip1Tg) mice. Fnip1Tg mice were crossed with Fnip1-knockout (Fnip1KO) mice to generate Fnip1TgKO mice expressing Fnip1 only in skeletal muscle but not in other tissues. Our results indicate that, in addition to the known role in type I fiber program, FNIP1 exerts control upon muscle mitochondrial oxidative program through AMPK signaling. Indeed, basal levels of FNIP1 are sufficient to inhibit AMPK but not mTORC1 activity in skeletal muscle cells. Gain-of-function and loss-of-function strategies in mice, together with assessment of primary muscle cells, demonstrated that skeletal muscle mitochondrial program is suppressed via the inhibitory actions of FNIP1 on AMPK. Surprisingly, the FNIP1 actions on type I fiber program is independent of AMPK and its downstream PGC-1α. These studies provide a vital framework for understanding the intrinsic role of FNIP1 as a crucial factor in the concerted regulation of mitochondrial function and muscle fiber type that determine muscle fitness.

TAK1 inhibition improves myoblast differentiation and alleviates fibrosis in a mouse model of Duchenne muscular dystrophy.

BACKGROUND: Transforming growth factor-ß-activated kinase 1 (TAK1) plays a key role in regulating fibroblast and myoblast proliferation and differentiation. However, the TAK1 changes associated with Duchenne muscular dystrophy (DMD) are poorly understood, and it remains unclear how TAK1 regulation could be exploited to aid the treatment of this disease. METHODS: Muscle biopsies were obtained from control donors or DMD patients for diagnosis (n = 6 per group, male, 2-3 years, respectively). Protein expression of phosphorylated TAK1 was measured by western blot and immunofluorescence analysis. In vivo overexpression of TAK1 was performed in skeletal muscle to assess whether TAK1 is sufficient to induce or aggravate atrophy and fibrosis. To explore whether TAK1 inhibition protects against muscle damage, mdx (loss of dystrophin) mice were treated with adeno-associated virus (AAV)-short hairpin TAK1 (shTAK1) or NG25 (a TAK1 inhibitor). Serum analysis, skeletal muscle performance and histology, muscle contractile function, and gene and protein expression were performed. RESULTS: We found that TAK1 was activated in the dystrophic muscles of DMD patients (n = 6, +72.2%, P < 0.001), resulting in fibrosis ( +65.9% for fibronectin expression, P < 0.001) and loss of muscle fibres (-32.5%, P < 0.01). Moreover, TAK1 was activated by interleukin-1ß, tumour necrosis factor-α, and transforming growth factor-ß1 (P < 0.01). Overexpression of TAK1 by AAV vectors further aggravated fibrosis (n = 8, +39.6% for hydroxyproline content, P < 0.01) and exacerbated muscle wasting (-31.6%, P < 0.01) in mdx mice; however, these effects were reversed in mdx mice by treatment with AAV-short hairpin TAK1 (shTAK1) or NG25 (a TAK1 inhibitor). The molecular mechanism underlying these effects may be related to the prevention of TAK1-mediated transdifferentiation of myoblasts into fibroblasts, thereby reducing fibrosis and increasing myoblast differentiation. CONCLUSIONS: Our findings show that TAK1 activation exacerbated fibrosis and muscle degeneration and that TAK1 inhibition can improve whole-body muscle quality and the function of dystrophic skeletal muscle. Thus, TAK1 inhibition may constitute a novel therapy for DMD.

Catalpol counteracts the pathology in a mouse model of Duchenne muscular dystrophy by inhibiting the TGF-ß1/TAK1 signaling pathway.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease caused by a mutation in the gene encoding the dystrophin protein. Catalpol is an iridoid glycoside found in Chinese herbs with anti-inflammatory, anti-oxidant, anti-apoptotic, and hypoglycemic activities that can protect against muscle wasting. In the present study we investigated the effects of catalpol on DMD. Aged Dystrophin-deficient (mdx) mice (12 months old) were treated with catalpol (100, 200 mg·kg-1·d-1, ig) for 6 weeks. At the end of the experiment, the mice were sacrificed, and gastrocnemius (GAS), tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL) muscles were collected. We found that catalpol administration dose-dependently increased stride length and decreased stride width in Gait test. Wire grip test showed that the time of wire grip and grip strength were increased. We found that catalpol administration dose-dependently alleviated skeletal muscle damage, evidenced by reduced plasma CK and LDH activity as well as increased the weight of skeletal muscles. Catalpol administration had no effect on dystrophin expression, but exerted anti-inflammatory effects. Furthermore, catalpol administration dose-dependently decreased tibialis anterior (TA) muscle fibrosis, and inhibited the expression of TGF-ß1, TAK1 and α-SMA. In primary myoblasts from mdx mice, knockdown of TAK1 abolished the inhibitory effects of catalpol on the expression levels of TGF-ß1 and α-SMA. In conclusion, catalpol can restore skeletal muscle strength and alleviate skeletal muscle damage in aged mdx mice, thus may provide a novel therapy for DMD. Catalpol attenuates muscle fibrosis by inhibiting the TGF-ß1/TAK1 signaling pathway.

A potential therapeutic effect of catalpol in Duchenne muscular dystrophy revealed by binding with TAK1.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by the loss of dystrophin, which results in inflammation, fibrosis, and the inhibition of myoblast differentiation in skeletal muscle. Catalpol, an iridoid glycoside, improves skeletal muscle function by enhancing myogenesis; it has potential to treat DMD. We demonstrate the positive effects of catalpol in dystrophic skeletal muscle. METHODS: mdx (loss of dystrophin) mice (n = 18 per group) were treated with catalpol (200 mg/kg) for six consecutive weeks. Serum analysis, skeletal muscle performance and histology, muscle contractile function, and gene and protein expression were performed. Molecular docking and ligand-target interactions, RNA interference, immunofluorescence, and plasmids transfection were utilized to explore the protective mechanism in DMD by which catalpol binding with transforming growth factor-ß-activated kinase 1 (TAK1) in skeletal muscle. RESULTS: Six weeks of catalpol treatment improved whole-body muscle health in mdx mice, which was characterized by reduced plasma creatine kinase (n = 18, -35.1%, P < 0.05) and lactic dehydrogenase (n = 18, -10.3%, P < 0.05) activity. These effects were accompanied by enhanced grip strength (n = 18, +25.4%, P < 0.05) and reduced fibrosis (n = 18, -29.0% for hydroxyproline content, P < 0.05). Moreover, catalpol treatment protected against muscle fatigue and promoted muscle recovery in the tibialis anterior (TA) and diaphragm (DIA) muscles (n = 6, +69.8%, P < 0.05 and + 74.8%, P < 0.001, respectively), which was accompanied by enhanced differentiation in primary myoblasts from DMD patients (n = 6, male, mean age: 4.7 ± 1.9 years) and mdx mice. In addition, catalpol eliminated p-TAK1 overexpression in mdx mice (n = 12, -21.3%, P < 0.05) and primary myoblasts. The catalpol-induced reduction in fibrosis and increased myoblast differentiation resulted from the inhibition of TAK1 phosphorylation, leading to reduced myoblast trans-differentiation into myofibroblasts. Catalpol inhibited the phosphorylation of TAK1 by binding to TAK1, possibly at Asp-206, Thr-208, Asn-211, Glu-297, Lys-294, and Tyr-293. CONCLUSIONS: Our findings show that catalpol and TAK1 inhibitors substantially improve whole-body muscle health and the function of dystrophic skeletal muscles and may provide a novel therapy for DMD.

Hypoglycaemic effect of catalpol in a mouse model of high-fat diet-induced prediabetes.

Type 2 diabetes mellitus is a major health problem and a societal burden. Individuals with prediabetes are at increased risk of type 2 diabetes mellitus. Catalpol, an iridoid glycoside, has been reported to exert a hypoglycaemic effect in db/db mice, but its effect on the progression of prediabetes is unclear. In this study, we established a mouse model of prediabetes and examined the hypoglycaemic effect, and the mechanism of any such effect, of catalpol. Catalpol (200 mg/(kg·day)) had no effect on glucose tolerance or the serum lipid level in a mouse model of impaired glucose tolerance-stage prediabetes. However, catalpol (200 mg/(kg·day)) increased insulin sensitivity and decreased the fasting glucose level in a mouse model of impaired fasting glucose/impaired glucose tolerance-stage prediabetes. Moreover, catalpol increased the mitochondrial membrane potential (1.52-fold) and adenosine triphosphate content (1.87-fold) in skeletal muscle and improved skeletal muscle function. These effects were mediated by activation of the insulin receptor-1/glucose transporter type 4 (IRS-1/GLUT4) signalling pathway in skeletal muscle. Our findings will facilitate the development of a novel approach to suppressing the progression of diabetes at an early stage. Novelty Catalpol prevents the progression of prediabetes in a mouse model of prediabetes. Catalpol improves insulin sensitivity in skeletal muscle. The effects of catalpol are mediated by activation of the IRS-1/GLUT4 signalling pathway.

Quantitative proteomics analysis of Fructus Psoraleae-induced hepatotoxicity in rats.

Fructus Psoraleae, which is commonly consumed for the treatment of osteoporosis, bone fracture, and leucoderma, induces liver injury. This study investigated the pathogenesis of the ethanol extract of Fructus Psoraleae (EEFP)-induced liver injury in rats. EEFP (1.35, 1.80, and 2.25 g·kg-1) was administrated to Sprague Dawley (SD) rats for 30 d. We measured liver chemistries, histopathology, and quantitative isobaric tags for relative and absolute quantitation (iTRAQ)-based protein profiling. EEFP demonstrated parameters suggestive of liver injury with changes in bile secretion, bile flow rate, and liver histopathology. iTRAQ analysis showed that a total of 4042 proteins were expressed in liver tissues of EEFP-treated and untreated rats. Among these proteins, 81 were upregulated and 32 were downregulated in the treatment group. KEGG pathway analysis showed that the drug metabolic pathways of cytochrome P450, glutathione metabolism, glycerolipid metabolism, and bile secretion were enriched with differentially expressed proteins. The expression of key proteins related to the farnesoid X receptor (FXR), i.e., the peroxisome proliferators-activated receptor alpha (PPAR-α), were downregulated, and multidrug resistance-associated protein 3 (MRP3) was upregulated in the EEFP-treated rats. Our results provide evidence that EEFP may induce hepatotoxicity through various pathways. Furthermore, our study demonstrates changes in protein regulation using iTRAQ quantitative proteomics analysis.

Imbalanced insulin substrate-1 and insulin substrate-2 signaling trigger hepatic steatosis in vitamin D deficient rats: 8-methoxypsoralen, a vitamin D receptor ligand with a promising anti-steatotic action.

The exact role of VD deficiency in the development of non-alcoholic fatty liver disease (NAFLD) remains unknown. In this study, we induced VD deficiency by feeding Female Sprague-Dawley rats a VD deficient (VDD) Diet and studied the hepatic changes associated with VD deficiency. Simultaneously, we provided the VDD rats with VD or 8-methoxy psoralen (8-MOP), a suggested vitamin D receptor agonist, to test the reversibility of the hepatic changes. VDD Rats developed borderline non-alcoholic steatohepatitis (NASH) with considerable elevation in hepatic triglycerides, total cholesterol, and malondialdehyde. Furthermore, VD deficiency induced the expression of crucial enzymes and transcription factors involved in denovo lipogenesis, which justified the hepatic lipid accumulation. Insulin receptor signaling was affected by VD deficiency, demonstrated by the elevation in insulin substrate-1 (IRS1) and reduction in insulin substrate-2 (IRS2) signaling. Treatment with VD or 8-MOP attenuated IRS1 signaling and its downstream targets, leading to a decline in de novo lipogenesis, while the elevation in IRS2 expression resulted in the nuclear exclusion of forkhead box O1 (FoxO1) and diminished gluconeogenesis, a vital source of acetyl-CoA for de novo lipogenesis. Moreover, 8-MOP and Calcipotriol modulated insulin signaling in human hepatocyte cell line L02, which highlighted the crucial role of VD in the regulation of hepatic lipid contents in rats and humans. Silencing of the vitamin D receptor expression in L02 diminished the inhibitory effect of Calcipotriol and 8-MOP on fatty acid synthase and acetyl- CoA carboxylase 1 and provided the evidence that 8-MOP actions mediated via vitamin D receptor.

The hypoglycemic mechanism of catalpol involves increased AMPK-mediated mitochondrial biogenesis.

Mitochondria serve as sensors of energy regulation and glucose levels, which are impaired by diabetes progression. Catalpol is an iridoid glycoside that exerts a hypoglycemic effect by improving mitochondrial function, but the underlying mechanism has not been fully elucidated. In the current study we explored the effects of catalpol on mitochondrial function in db/db mice and C2C12 myotubes in vitro. After oral administration of catalpol (200 mg·kg-1·d-1) for 8 weeks, db/db mice exhibited a decreased fasting blood glucose level and restored mitochondrial function in skeletal muscle. Catalpol increased mitochondrial biogenesis, evidenced by significant elevations in the number of mitochondria, mitochondrial DNA levels, and the expression of three genes associated with mitochondrial biogenesis: peroxisome proliferator-activated receptor gammaco-activator 1 (PGC-1α), mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF1). In C2C12 myotubes, catalpol significantly increased glucose uptake and ATP production. These effects depended on activation of AMP-activated protein kinase (AMPK)-mediated mitochondrial biogenesis. Thus, catalpol improves skeletal muscle mitochondrial function by activating AMPK-mediated mitochondrial biogenesis. These findings may guide the development of a new therapeutic approach for type 2 diabetes.

Evaluating supersaturation in vitro and predicting its performance in vivo with Biphasic gastrointestinal Simulator: A case study of a BCS IIB drug.

This study aimed to develop an evaluation approach for supersaturation by employing an in vitro bio-mimicking apparatus designed to predict in vivo performance. The Biphasic Gastrointestinal Simulator (BGIS) is composed of three chambers with absorption phases that represent the stomach, duodenum, and jejunum, respectively. The concentration of apatinib in each chamber was detected by fiber optical probes in situ. The dissolution data and the pharmacokinetic data were correlated by GastroplusTM. The precipitates were characterized by polarizing microscope, Scanning Electron Microscopy, Powder X-ray diffraction and Differential scanning calorimetry. According to the results, Vinylpyrrolidone-vinyl acetate copolymer (CoPVP) prolonged supersaturation by improving solubility and inhibiting crystallization, while Hydroxypropyl methylcellulose (HPMC) prolonged supersaturation by inhibiting crystallization alone. Furthermore, a predictive in vitro-in vivo correlation was established, which confirmed the anti-precipitation effect of CoPVP and HPMC on in vitro performance and in vivo behavior. In conclusion, CoPVP and HPMC increased and prolonged the supersaturation of apatinib, and then improved its bioavailability. Moreover, BGIS was demonstrated to be a significant approach for simulating in vivo conditions for in vitro-in vivo correlation in a supersaturation study. This study presents a promising approach for evaluating supersaturation, screening precipitation inhibitors in vitro, and predicting their performances in vivo.

Mitochondria-dependent apoptosis in triptolide-induced hepatotoxicity is associated with the Drp1 activation.

How triptolide is associated with mitochondrial dysfunction and apoptosis in connection with its hepatotoxicity remains unclear. The objective of our study was to find out the link between mitochondrial dynamics and cell death in triptolide induced hepatotoxicity. We treated L02 cells with 25 nM concentration of triptolide. The results demonstrated that triptolide treatment caused an increase in apoptotic cell death, mitochondrial depolarization, ROS overproduction, a decrease in ATP production, and mitochondrial fragmentation which in turn is associated with the activation of Drp1 fission protein. Triptolide treatment led to the translocation of Drp1 from the cytosol into outer mitochondrial membrane where it started mitochondrial fission. This fission event is coupled with the mitochondrial release of cytochrome c into the cytosol and subsequently caspase-3 activation. TEM analysis of rat liver tissues revealed the distortion of mitochondrial morphology in triptolide-treated group. Western blot analysis explained that disruption in mitochondrial morphology was attached with the recruitment of Drp1 to mitochondria, cytochrome c release, and caspase-3 activation. However, Mdivi-1 co-treatment inhibited the activation of Drp1 and caspase-3 and blocked the release of cytochrome c into the cytosol. In short, inhibiting Drp1 protein activation may provide a new potential target for curing Drp1-associated apoptosis in triptolide-induced hepatotoxicity.

Key role of organic cation transporter 2 for the nephrotoxicity effect of triptolide in rheumatoid arthritis.

Tripterygium wilfordii Hook. F. (TwHF), a traditional Chinese Medicine, is effective in treating rheumatoid arthritis (RA), but its severe nephrotoxicity limits its extensive application. The nephrotoxic mechanism of Triptolide (TP), the main pharmacological and toxic component of TwHF, has not been fully revealed. This study was designed to explore the nephrotoxicity of TP in the RA state and the potential molecular mechanism. A rat collagen-induced arthritis (CIA) model was constructed and administered with TP for 28 days in vivo. Results showed that the kidney injury induced by TP was aggravated in the CIA state, the concentration of TP in the renal cortex was higher than that of the medulla after TP administration in the CIA rats, and the expression of organic cation transporter 2 (Oct2) in kidney was up-regulated under CIA condition. Besides, rat kidney slice study demonstrated that TP was transported by Oct2 and this was confirmed by transient silencing and overexpression of OCT2 in HEK-293T cells. Furthermore, cytoinflammatory models on HK-2 and HEK-293T cell lines were constructed by exposure of TNF-α or IL-1ß to further explore the TP's renal toxicity. Results suggested that TNF-α exposure aggravated TP's toxicity and up-regulated the protein expression of OCT2 in both cell lines. TNF-α treatment also increased the function of OCT2 and finally OCT2 silencing confirmed OCT2 mediated nephrotoxicity of TP in HEK-293T cells. In summary, the exposure of TNF-α in RA state induced the expression of OCT2, which transported more TP into kidney cortex, subsequently exacerbated the kidney injury.