Poricoic acid a ameliorates high glucose-induced podocyte injury by regulating the AMPKα/FUNDC1 pathway.

BACKGROUND: Poricoic acid A (PAA), a major triterpenoid component of Poria cocos with anti-tumor, anti-fibrotic, anti-inflammatory, and immune-regulating activities, has been shown to induce podocyte autophagy in diabetic kidney disease (DKD) by downregulating FUN14 domain containing 1 (FUNDC1). This study aimed to identify the role of adenosine monophosphate-activated protein kinase alpha (AMPKα) in PAA-mediated phosphorylation of FUNDC1 in podocyte injury occurring in the pathogenesis of DKD. METHODS AND RESULTS: A cellular model of renal podocyte injury was established by culturing MPC5 cells under high-glucose (HG) conditions. MPC5 cells were subjected to transfection with small interfering RNA (siRNA) targeting AMPKα or siRNA targeting FUNDC1, an AMPKα activator, or PAA. PAA treatment induced the phosphorylation of AMPKα in HG-cultured podocytes. AMPKα activation was implicated in the inhibitory effect of PAA on FUNDC phosphorylation in HG-cultured podocytes. Treatment targeting the AMPKα activator also significantly augmented proliferation, migration, mitochondrial membrane potential, and autophagy levels, while reducing apoptosis levels, inhibiting oxidative stress, and suppressing the release of proinflammatory factors in HG-cultured MPC5 cells. In contrast, insufficient expression of AMPKα reversed the effects of PAA on the proliferation, migration, and apoptosis of podocytes and further exacerbated the reduction of phosphorylated FUNDC1 expression in podocytes under HG conditions. CONCLUSIONS: AMPKα is involved in the regulation of FUNDC1 phosphorylation by PAA in HG-induced podocyte injury. Furthermore, the AMPKα/FUNDC1 pathway plays a crucial regulatory role in HG-induced podocyte injury. These findings support AMPKα, FUNDC1, and the AMPKα/FUNDC1 pathway as targets for PAA intervention.

Poricoic acid A induces mitophagy to ameliorate podocyte injury in diabetic kidney disease via downregulating FUNDC1.

Diabetic kidney disease (DKD) is a devastating complication of diabetes mellitus (DM) and is the most prevalent chronic kidney disease (CKD). Poricoic acid A (PAA), a component isolated from Traditional Chinese Medicine (TCM) Poria cocos, has hypoglycaemic and anti-fibrosis effects. However, the role of PAA in DKD remains largely unclear. To mimics an in vitro model of DKD, the mouse podocyte MPC5 cells were treated with high glucose (25 mM; HG) for 24 h. CCK-8 and flow cytometry assays were conducted for assessing MPC5 cell viability and apoptosis. Meanwhile, streptozotocin (STZ) was used to induce experimental DKD in mice by intraperitoneal injection. PAA notably inhibited the apoptosis and inflammation, reduced the generation of ROS, and elevated the MMP level in HG-treated MPC5 cells. Moreover, PAA obviously reduced blood glucose and urine protein levels, inhibited renal fibrosis in DKD mice. Meanwhile, PAA markedly increased LC3 and ATG5 levels and declined p62 and FUNDC1 levels in HG-treated MPC5 cells and in the kidney tissues of DKD mice, leading to the activation of cell mitophagy. Furthermore, the downregulation of FUNDC1 also inhibited apoptosis, inflammation, and promoted mitophagy in HG-treated MPC5 cells. As expected, the knockdown of FUNDC1 further enhanced the protective role of PAA in MPC5 cells following HG treatment, indicating that induction of mitophagy could attenuate podocyte injury. Collectively, PAA could exert beneficial effects on podocyte injury in DKD by promoting mitophagy via downregulating FUNDC1. These findings suggested that PAA may have great potential in alleviating kidney injury in DKD.

Poricoic acid A suppresses renal fibroblast activation and interstitial fibrosis in UUO rats via upregulating Sirt3 and promoting ß-catenin K49 deacetylation.

Renal interstitial fibrosis is the common pathological process of various chronic kidney diseases to end-stage renal disease. Inhibition of fibroblast activation attenuates renal interstitial fibrosis. Our previous studies show that poricoic acid A (PAA) isolated from Poria cocos is a potent anti-fibrotic agent. In the present study we investigated the effects of PAA on renal fibroblast activation and interstitial fibrosis and the underlying mechanisms. Renal interstitial fibrosis was induced in rats or mice by unilateral ureteral obstruction (UUO). UUO rats were administered PAA (10 mg·kg-1·d-1, i.g.) for 1 or 2 weeks. An in vitro model of renal fibrosis was established in normal renal kidney fibroblasts (NRK-49F cells) treated with TGF-ß1. We showed that PAA treatment rescued Sirt3 expression, and significantly attenuated renal fibroblast activation and interstitial fibrosis in both the in vivo and in vitro models. In TGF-ß1-treated NRK-49F cells, we demonstrated that Sirt3 deacetylated ß-catenin (a key transcription factor of fibroblast activation) and then accelerated its ubiquitin-dependent degradation, thus suppressing the protein expression and promoter activity of pro-fibrotic downstream target genes (twist, snail1, MMP-7 and PAI-1) to alleviate fibroblast activation; the lysine-49 (K49) of ß-catenin was responsible for Sirt3-mediated ß-catenin deacetylation. In molecular docking analysis, we found the potential interaction of Sirt3 and PAA. In both in vivo and in vitro models, pharmacological activation of Sirt3 by PAA significantly suppressed renal fibroblast activation via facilitating ß-catenin K49 deacetylation. In UUO mice and NRK-49F cells, Sirt3 overexpression enhanced the anti-fibrotic effect of PAA, whereas Sirt3 knockdown weakened the effect. Taken together, PAA attenuates renal fibroblast activation and interstitial fibrosis by upregulating Sirt3 and inducing ß-catenin K49 deacetylation, highlighting Sirt3 functions as a promising therapeutic target of renal fibroblast activation and interstitial fibrosis.

Poricoic acid A (PAA) inhibits T-cell acute lymphoblastic leukemia through inducing autophagic cell death and ferroptosis.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer with poor clinical outcome. Poricoic acid A (PAA) is the main chemical constituent on the surface layer of the mushroom Poria cocos, and exerts protective effects against various diseases. In the study, its effects on T-ALL progression were investigated both in vitro and in vivo. Our results showed that PAA strongly reduced the cell viability of T-ALL cell lines, and induced cell G2 cycle arrest and apoptosis in vitro. Mitochondrial dysfunction was also elevated by PAA, along with enhanced cellular reactive oxygen species (ROS) production. Importantly, PAA-suppressed cell viability and -triggered apoptosis were ROS-dependent. Additionally, autophagy was significantly induced by PAA in T-ALL cells through regulating AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) and LC3 signaling pathways. PAA treatments also provoked ferroptosis in T-ALL cells with reduced glutathione (GSH) levels and elevated malonaldehyde (MDA) contents. Suppressing autophagy and ferroptosis almost abrogated the capacity of PAA to restrain T-ALL proliferation and growth. The effects of PAA to suppress T-ALL tumor growth were also confirmed in vivo with undetectable toxicity. Therefore, the present study highlighted the potential of PAA for T-ALL treatment mainly through inducing autophagic cell death and ferroptosis.

Poricoic acid A promotes angiogenesis and myocardial regeneration by inducing autophagy in myocardial infarction.

Myocardial infarction (MI) is a kind of cardiovascular diseases with high morbidity and mortality. Poricoic acid A (PAA) is the main active substance in Poria cocos, which has been discovered to exhibit an ameliorative role in the progression of many diseases. However, no report has been focused on the regulatory effects of PAA on MI progression. In this study, at first, oxygen glucose deprivation (OGD) treatment was performed in human cardiac microvascular endothelial cells (HCMECs) to mimic MI cell model. Our findings demonstrated that cell proliferation was reduced post OGD treatment, but which was reversed by PAA treatment. Moreover, PAA suppressed cell apoptosis in OGD-triggered HCMEC cells. Next, it revealed that PAA induced autophagy in OGD-treated HCMEC cells through enhancing LC3-II/LC3-I level and reducing P62 level. In addition, PAA strengthened the angiogenesis ability and migration ability in OGD-induced HCMEC cells. Lastly, it was uncovered that PAA modulated the AMPK/mTOR signaling pathway through affecting the p-mTOR/mTOR and p-AMPK/AMPK levels. In conclusion, PAA can promote angiogenesis and myocardial regeneration after MI by inducing autophagy through modulating the AMPK/mTOR pathway. This work suggested that PAA may be a potential and useful drug for MI treatment.

Poricoic acid A suppresses TGF-ß1-induced renal fibrosis and proliferation via the PDGF-C, Smad3 and MAPK pathways.

Renal interstitial fibrosis is the most important pathological process in chronic renal failure. Previous studies have shown that poricoic acid A (PAA), the main chemical constituent on the surface layer of the mushroom Poria cocos, has protective effects against oxidative stress and acute kidney injury. The present study aimed to investigate the potential roles of PAA on the pathological process of renal fibrosis and the associated molecular mechanism. The NRK-49F cell line was treated with transforming growth factor-ß1 (TGF-ß1) with or without PAA or platelet-derived growth factor C (PDGF-C). Cell Counting Kit-8 assay, western blotting and 5-ethynyl-2'-deoxyuridine immunofluorescence staining were performed to examine cell growth, protein expression and cell proliferation, respectively. Data from the present study showed that 10 µM PAA attenuated TGF-ß1-induced NRK-49F cell extracellular matrix (ECM) accumulation, fibrosis formation and proliferation. Renal fibrosis with the activation of Smad3 and mitogen-activated protein kinase (MAPK) pathways were also inhibited by PAA treatment. PDGF-C reversed the inhibitory effects of PAA on TGF-ß1-induced renal fibroblast proliferation and activation of the Smad3/MAPK pathway. The present study suggested that suppression of TGF-ß1-induced renal fibroblast ECM accumulation, fibrosis formation and proliferation by PAA is mediated via the inhibition of the PDGF-C, Smad3 and MAPK pathways. The present findings not only revealed the potential anti-fibrotic effects of PAA on renal fibroblasts, but also provided a new insight into the prevention of fibrosis formation via regulation of the PDGF-C, Smad3 and MAPK signaling pathways.

Poricoic acid A as a modulator of TPH-1 expression inhibits renal fibrosis via modulating protein stability of ß-catenin and ß-catenin-mediated transcription.

BACKGROUND: Renal fibrosis is the common feature of chronic kidney disease (CKD). However, few drugs specifically target fibrogenesis due to the lack of an effective therapeutic target. Hence, it is urgent to find a therapeutic strategy that inhibits renal fibrosis. Here, we identified that poricoic acid A (PAA) as the modulator of tryptophan hydroxylase-1 (TPH-1), the key enzyme in tryptophan metabolism, exerted potent anti-fibrotic effects in the kidney. METHODS: Lentiviral vector, luciferase reporter activity assay and co-immunoprecipitation were used. The animal model of unilateral ureteral obstruction and adenine-induced chronic renal failure as well as transforming growth factor (TGF)-ß1-treated epithelial cells NRK-52E and fibroblasts NRK-49F were used. RESULTS: TPH-1 was gradually decreased during CKD progression, while PAA treatment significantly increased TPH-1 expression to suppress renal fibrosis. Pharmacological overexpression of TPH-1 by PAA treatment exhibited anti-fibrosis and was linked to Wnt/ß-catenin signaling activity. TPH-1 exhibited anti-fibrotic effects by suppressing epithelial cell injury and fibroblast activation, and PAA promoted TPH-1 expression and then suppressed the Wnt/ß-catenin signaling pathway via regulating the protein stability of ß-catenin and ß-catenin-mediated transcription. TPH-1 overexpression enhanced the anti-fibrotic effects of PAA, while TPH-1 deficiency weakened the anti-fibrotic effects of PAA, indicating that TPH-1 was required for the anti-fibrotic effects of PAA. CONCLUSION: PAA as a modulator of TPH-1 expression attenuated renal fibrosis through regulating the Wnt/ß-catenin signaling pathway by acting on the protein stability of ß-catenin and ß-catenin-mediated transcription. TPH-1 was required for PAA to exert anti-fibrosis.

Poricoic acid A activates AMPK to attenuate fibroblast activation and abnormal extracellular matrix remodelling in renal fibrosis.

BACKGROUND: In chronic kidney disease, although fibrosis prevention is beneficial, few interventions are available that specifically target fibrogenesis. Poricoic acid A (PAA) isolated from Poria cocos exhibits anti-fibrotic effects in the kidney, however the underlying mechanisms remain obscure. PURPOSE: We isolated PAA and investigated its effects and the underlying mechanisms in renal fibrosis. STUDY DESIGN: Unilateral ureteral obstruction (UUO) and 5/6 nephrectomy (Nx) animal models and TGF-ß1-induced renal fibroblasts (NRK-49F) were used to investigate the anti-fibrotic activity of PAA and its underlying mechanisms. METHODS: Western blots, qRT-PCR, immunofluorescence staining, co-immunoprecipitation and molecular docking methods were used. Knock-down and knock-in of adenosine monophosphate-activated protein kinase (AMPK) in the UUO model and cultured NRK-49F cells were employed to verify the mechanisms of action of PAA. RESULTS: PAA improved renal function and alleviated fibrosis by stimulating AMPK and inhibiting Smad3 specifically in Nx and UUO models. Reduced AMPK activity was associated with Smad3 induction, fibroblast activation, and the accumulation and aberrant remodelling of extracellular matrix (ECM) in human renal puncture samples and cultured NRK-49F cells. PAA stimulated AMPK activity and decreased fibrosis in a dose-dependent manner, thus showing that AMPK was essential for PAA to exert its anti-fibrotic effects. AMPK deficiency reduced the anti-fibrotic effects of PAA, while AMPK overexpression enhanced its effect. CONCLUSION: PAA activated AMPK and further inhibited Smad3 specifically to suppress fibrosis by preventing aberrant ECM accumulation and remodelling and facilitating the deactivation of fibroblasts.

Subunit-specific effects of poricoic acid A on NMDA receptors.

BACKGROUND: N-methyl-D-aspartate (NMDA) receptor is a tetrameric protein complex composed of glycine-linked NR1 subunits and glutamate-linked NR2 subunits. There are four NR2 subunits (A-D) that differ in development, anatomy, and function profiles. They play various roles in normal and neuropathologic conditions. Specific agonists, antagonists, and modulators of subunits for selective NMDA receptors may be precious mediational tools and potent agents for treating diseases. The objective of this study was to determine the effect of poricoic acid A on NMDA receptor known to mediate excitatory synaptic transmission factors and cause changes in synaptic strength. Inhibitory effect of poricoic acid A on NR1a combined with NR2A, NR2B, NR2C, or NR2D receptor was evaluated. METHODS: Glutamate-mediated currents for each NR1a and NR2 subunits were investigated using two-electrode voltage-clamp techniques. Molecular modeling and molecular dynamics simulation studies were carried out with Autodock Tools. Poricoic acid A and NMDA receptor protein complex were examined with Ligplot and Pymol docking program. Ligplot shows binding activity at the protein and the ligand. RESULTS: The inhibitory effect of poricoic acid A on glutamate-induced inward current in a concentration-dependent manner that was reversible. Half inhibitory concentrations of glutamate on NR1a/NR2A, NR1a/NR2B, NR1a/NR2C, and NR1a/NR2D receptors were 9.6 ± 1.2, 5.7 ± 0.4, 46.1 ± 21.5, and 21.5 ± 8.2 µM, respectively. This corresponded to the order of inhibitory effect of oocyte expressing NR1a and NR2s subunit of NR1a/NR2B > NR1a/NR2A > NR1a/NR2C > NR1a/NR2D. CONCLUSIONS: Taken together, these results indicate that poricoic acid A can modulate the expression of NMDA receptor. In addition, the regulation of excitatory ligand-gating ion channel by poricoic acid A may have pharmaceutical functions on excitatory synaptic transmission of neuronal system.

Comparison and quantitative analysis of wild and cultivated Macrohyporia cocos using attenuated total refection-Fourier transform infrared spectroscopy combined with ultra-fast liquid chromatography.

Dried sclerotium of Macrohyporia cocos is a well-known and widely-consumed traditional Chinese medicine and is also used as dietary supplement. According to the differential treatment between cultivation and wild habitats in the market, the comparison and quantitative analysis of wild and cultivated M. cocos were performed using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and ultra-fast liquid chromatography combined with partial least squares discriminant analysis and partial least squares regression (PLSR). 636 samples were used for the spectral scan and chromatographic analysis. Results indicated that contents of dehydrotumulosic acid, poricoic acid A and dehydrotrametenolic acid in cultivated samples were significantly different from wild samples in two medicinal parts. Differences of dehydropachymic acid and pachymic acid just existed in inner part samples (P < 0.05). Wild M. cocos samples could be discriminated with cultivated samples with >95.14% efficiency using spectral data. ATR-FTIR combined with PLSR provided satisfactory performance for content predictions of poricoic acid A and dehydrotrametenolic acid. This study demonstrated that growth patterns could affect the quality of inner part and epidermis of M. cocos, and ATR-FTIR was a promising technique for the identification of wild and cultivated M. cocos and the rapid determination of triterpene acids contents.

Poricoic acid A enhances melatonin inhibition of AKI-to-CKD transition by regulating Gas6/AxlNFκB/Nrf2 axis.

Renal ischemia-reperfusion injury (IRI) is a complex syndrome, which causes chronic kidney disease (CKD) after recovery from IRI-mediated acute kidney injury (AKI). There is no single therapy that could effectively prevent the renal injury after ischemia. In this study, the effects of melatonin or poricoic acid A (PAA) and their combination were investigated in protecting against AKI-to-CKD transition in rats and hypoxia/reoxygenation (H/R)-induced injury in cultured renal NRK-52E cells. Melatonin and PAA significantly reduced the magnitude of rise in serum creatinine and urea levels in IRI rats at days 3 and 14. Our results further showed that treatment with melatonin and PAA ameliorated renal fibrosis and podocyte injury by attenuating oxidative stress and inflammation via regulation of nuclear factor-kappa B (NF-κB) and nuclear factor-erythroid-2-related factor 2 (Nrf2) pathways in IRI rats. Melatonin and PAA protected against AKI-to-CKD transition by regulating growth arrest-specific 6 (Gas6)/AxlNFκB/Nrf2 signaling cascade. Melatonin and PAA initiallyupregulated Gas6/Axl signaling to reduce oxidative stress and inflammation in AKI and subsequently downregulated Gas6/Axl signaling to attenuate renal fibrosis and progression to CKD. Melatonin and PAA inhibited expression of extracellular matrix proteins. Poricoic acid A enhances melatonin-mediated inhibition of AKI-to-CKD transition by the regulating Gas6/AxlNFκB/Nrf2 signaling cascade. Notably, our study first identified Axl as a promising therapeutic target for prevention of AKI-to-CKD transition.

Combined melatonin and poricoic acid A inhibits renal fibrosis through modulating the interaction of Smad3 and ß-catenin pathway in AKI-to-CKD continuum.

BACKGROUND: Acute kidney injury (AKI) is one of the major risk factors for progression to chronic kidney disease (CKD) and renal fibrosis. However, effective therapies remain poorly understood. Here, we examined the renoprotective effects of melatonin and poricoic acid A (PAA) isolated from the surface layer of Poria cocos, and investigated the effects of combined therapy on the interaction of TGF-ß/Smad and Wnt/ß-catenin in a rat model of renal ischemia-reperfusion injury (IRI) and hypoxia/reoxygenation (H/R) or TGF-ß1-induced HK-2 cells. METHODS: Western blot and immunohistochemical staining were used to examine protein expression, while qRT-PCR was used to examine mRNA expression. Coimmunoprecipitation, chromatin immunoprecipitation, RNA interference, and luciferase reporter gene analysis were employed to explore the mechanisms of PAA and melatonin's renoprotective effects. RESULTS: PAA and combined therapy exhibited renoprotective and antifibrotic effects, but the underlying mechanisms were different during AKI-to-CKD continuum. Melatonin suppressed Smad-dependent and Smad-independent pathways, while PAA selectively inhibited Smad3 phosphorylation through distrupting the interactions of Smad3 with TGFßRI and SARA. Further studies demonstrated that the inhibitory effects of melatonin and PAA were partially depended on Smad3, especially PAA. Melatonin and PAA also inhibited the Wnt/ß-catenin pathway and its profibrotic downstream targets, and PAA performed better. We further determined that IRI induced a nuclear Smad3/ß-catenin complex, while melatonin and PAA disturbed the interaction of Smad3 and ß-catenin, and supplementing with PAA could enhance the inhibitory effects of melatonin on the TGF-ß/Smad and Wnt/ß-catenin pathways. CONCLUSIONS: Combined melatonin and PAA provides a promising therapeutic strategy to treat renal fibrosis during the AKI-to-CKD continuum.