Gubi Zhitong formula alleviates osteoarthritis in vitro and in vivo via regulating BNIP3L-mediated mitophagy.

BACKGROUND: Osteoarthritis (OA) is characterized by degeneration of articular cartilage, leading to joint pain and dysfunction. Gubi Zhitong formula (GBZTF), a traditional Chinese medicine formula, has been used in the clinical treatment of OA for decades, demonstrating definite efficacy. However, its mechanism of action remains unclear, hindering its further application. METHODS: The ingredients of GBZTF were analyzed and performed with liquid chromatography-mass spectrometry (LC-MS). 6 weeks old SD rats were underwent running exercise (25 m/min, 80 min, 0°) to construct OA model with cartilage wear and tear. It was estimated by Micro-CT, Gait Analysis, Histological Stain. RNA-seq technology was performed with OA Rats' cartilage, and primary chondrocytes induced by IL-1ß (mimics OA chondrocytes) were utilized to evaluated and investigated the mechanism of how GBZTF protected OA cartilage from being damaged with some functional experiments. RESULTS: A total of 1006 compounds were identified under positive and negative ion modes by LC-MS. Then, we assessed the function of GBZTF through in vitro and vivo. It was found GBZTF could significantly up-regulate OA rats' limb coordination and weight-bearing capacity, and reduce the surface and sub-chondral bone erosions of OA joints, and protect cartilage from being destroyed by inflammatory factors (iNOS, IL-6, IL-1ß, TNF- α, MMP13, ADAMTS5), and promote OA chondrocytes proliferation and increase the S phage of cell cycle. In terms of mechanism, RNA-seq analysis of cartilage tissues revealed 1,778 and 3,824 differentially expressed genes (DEGs) in model vs control group and GBZTF vs model group, respectively. The mitophagy pathway was most significantly enriched in these DEGs. Further results of subunits of OA chondrocytes confirmed that GBZTF could alleviate OA-associated inflammation and cartilage damage through modulation BCL2 interacting protein 3-like (BNIP3L)-mediated mitophagy. CONCLUSION: The therapeutic effectiveness of GBZTF on OA were first time verified in vivo and vitro through functional experiments and RNA-seq, which provides convincing evidence to support the molecular mechanisms of GBZTF as a promising therapeutic decoction for OA.

Fangji Huangqi decoction ameliorates membranous nephropathy through the upregulation of BNIP3-mediated mitophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Fangji Huangqi Decoction (FJHQ), a traditional Chinese medicinal formula outlined in Zhang Zhongjing's "Jin Gui Yao Lue" during the Han Dynasty, is often used to treat conditions characterized by symptoms like edema and dysuria, including membranous nephropathy (MN). Despite its proven clinical effectiveness, the exact mechanisms through which FJHQ acts on MN remain elusive. AIM OF THE STUDY: This study aimed to investigate whether FJHQ enhances BNIP3-mediated mitophagy in podocytes by promoting BNIP3 expression and whether this improvement leads to the amelioration of MN. MATERIALS AND METHODS: In this study, by establishing passive Heymann nephritis (PHN) rats, an experimental rat model of MN induced by sheep anti-rat Fx1A serum, we evaluated the effects of FJHQ in vivo. In vitro experiments were carried out by treating primary podocytes with experimental rat serum. Furthermore, the potential mechanism by which FJHQ acts through BNIP3 was further examined by transfecting primary podocytes with the siRNA of BNIP3 or the corresponding control vector. RESULTS: After 4 weeks, significant kidney damage was observed in the rats in the model group, comparatively, FJHQ markedly decreased urine volume, 24-h urinary protein, blood urea nitrogen (BUN), creatinine (Scr), and increased serum total albumin (ALB). Histology showed that FJHQ caused significant improvements in glomerular hyperplasia, and IgG immune complex deposition in MN rats. JC-1 fluorescence labelling and flow cytometry analysis showed that FJHQ could significantly increase mitochondrial membrane potential in vivo. In the mitochondria of MN model rats, FJHQ was able to down-regulate the expression of P62 and up-regulate the expression of BNIP3, LC3B, and LC3 II/LC3 I, according to Western blot and immunofluorescence studies. Furthermore, FJHQ has been shown to significantly up-regulate mitochondrial membrane potential, down-regulate P62 expression in mitochondria, and up-regulate the expression of BNIP3, LC3B, and LC3 II/LC3 I in mitochondria at the cellular level. After the administration of the autophagy inhibitor chloroquine, the serum of rats treated with FJHQ further increased the expression of LC3 II/LC3 I in primary podocytes, showing higher autophagy flow. After the interference of BNIP3 in podocytes, the effect of FJHQ on mitochondrial membrane potential and autophagy-related proteins almost disappeared. CONCLUSION: FJHQ enhanced mitophagy in podocytes by promoting the expression of BNIP3, thereby contributing to the amelioration of MN. This work reveals the possible underlying mechanism by which FJHQ improves MN and provides a new avenue for MN treatment.

Role of the HIF-1α/BNIP3 Signaling Pathway in Recurrent Hepatocellular Carcinoma and the Mechanism of Traditional Chinese Medicine.

Recurrence of hepatocellular carcinoma (HCC) negatively affects the quality of life of patients and leads to death. Studies have shown that recurrent hepatocellular carcinoma (RHCC) is closely related to tissue hypoxia and autophagy. It has been shown that hypoxia-inducible factor-1α (HIF-1α) and its downstream factor BCL-2 19 kDa-interacting protein 3 (BNIP3) promote cellular autophagy under hypoxic conditions, resulting in metastasis and RHCC. In this article, the molecular structures of HIF-1α and BNIP3 are described, and the significance of the HIF-1α/BNIP3 signaling pathway in RHCC is explained. Moreover, the role and mechanism of traditional Chinese medicine (TCM) in treating RHCC by modulating the HIF-1α/BNIP3 signaling pathway is discussed. Studies have shown that the HIF-1α/BNIP3 signaling pathway is a potential target of TCM in the treatment of RHCC. The mechanism of the HIF-1α/BNIP3 signaling pathway in RHCC and the progress achieved in TCM research on targeting and regulating this pathway are also reviewed in this article. The objective was to provide a theoretical basis for the prevention and treatment of RHCC, as well as further drug development.

Desferoxamine protects against hemophilic arthropathy through the upregulation of HIF-1α-BNIP3 mediated mitophagy.

AIMS: Hemophilic arthropathy (HA) is a typically iron overload induced joint disease secondary to continuous joint bleeding, however, the exact role of iron chelators in HA has not been fully elucidated. In the present study, we investigated whether desferoxamine (DFO), an iron chelator, could limit the development of HA and the underlying mechanisms. MATERIALS AND METHODS: A HA mice model was established by needle puncture in the left knees of FVIII-deficient hemophilic mice. HA progression was evaluated at 8 weeks after DFO administration. Moreover, chondrocytes were treated with ferric ammonium citrate (FAC) to mimic iron overload in vitro. Modulating effect of DFO on iron overload induced oxidative stress, chondrocytes apoptosis and extracellular matrix (ECM) degradation and the role of HIF-1α-BNIP3 mediated mitophagy were examined. KEY FINDINGS: We found that DFO limited the development of HA and protected iron overload induced ECM degradation, chondrocytes apoptosis and oxidative stress. Besides chelating Fe2+, we found that HIF-1α-BNIP3 mediated mitophagy played important roles in the protective effect of DFO. HIF-1α inhibition suppressed chondrocytes mitophagy process and partly diminished the protective effect of DFO on chondrocytes iron overload. SIGNIFICANCE: In conclusion, DFO could protect against HA development via HIF-1α-BNIP3 mediated mitophagy, suggesting DFO might be a potential therapeutic supplement for HA treatment.

The involvement of Parkin-dependent mitophagy in the anti-cancer activity of Ginsenoside.

Colon cancer, the third most frequent occurred cancer, has high mortality and extremely poor prognosis. Ginsenoside, the active components of traditional Chinese herbal medicine Panax ginseng, exerts antitumor effect in various cancers, including colon cancer. However, the detailed molecular mechanism of Ginsenoside in the tumor suppression have not been fully elucidated. Here, we chose the representative ginsenoside Rg3 and reported for the first time that Rg3 induces mitophagy in human colon cancer cells, which is responsible for its anticancer effect. Rg3 treatment leads to mitochondria damage and the formation of mitophagosome; when autophagy is inhibited, the clearance of damaged mitochondria can be reversed. Next, our results showed that Rg3 treatment activates the PINK1-Parkin signaling pathway and recruits Parkin and ubiquitin proteins to mitochondria to induce mitophagy. GO analysis of Parkin targets showed that Parkin interacts with a large number of mitochondrial proteins and regulates the molecular function of mitochondria. The cellular energy metabolism enzyme GAPDH is validated as a novel substrate of Parkin, which is ubiquitinated by Parkin. Moreover, GAPDH participates in the Rg3-induced mitophagy and regulates the translocation of Parkin to mitochondria. Functionally, Rg3 exerts the inhibitory effect through regulating the nonglycolytic activity of GAPDH, which could be associated with the cellular oxidative stress. Thus, our results revealed GAPDH ubiquitination by Parkin as a crucial mechanism for mitophagy induction that contributes to the tumor-suppressive function of ginsenoside, which could be a novel treatment strategy for colon cancer.

Hydroxysafflor Yellow A Alleviates Ischemic Stroke in Rats via HIF-1[Formula: see text], BNIP3, and Notch1-Mediated Inhibition of Autophagy.

Stroke has become a major cause of death and disability worldwide. The cellular recycling pathway autophagy has been implicated in ischemia-induced neuronal changes, but whether autophagy plays a beneficial or detrimental role is controversial. Hydroxysafflor Yellow A (HSYA), a popular herbal medicine, is an extract of Carthamus tinctorius and is used to treat ischemic stroke (IS) in China. HSYA has been shown to prevent cardiovascular and cerebral ischemia/reperfusion injury in animal models. However, the specific active ingredients and molecular mechanisms of HSYA in IS remain unclear. Here, we investigated the effect of HSYA treatment on autophagy in a rat model of IS. IS was induced in rats by middle cerebral artery occlusion. Rats were treated once daily for 3 days with saline, HYSA, or the neuroprotective agent Edaravone. Neurobehavioral testing was performed on days 1, 2, and 3 post-surgery. Brains were removed on day 3 post-surgery for histological evaluation of infarct area, morphology, and for qRT-PCR and western blot analysis of the expression of the autophagy factor LC3 and the signaling molecules HIF-1[Formula: see text], BNIP3, and Notch1. Molecular docking studies were performed in silico to predict potential interactions between HSYA and LC3, HIF-1[Formula: see text], BNIP3, and Notch1 proteins. The result showed that HSYA treatment markedly alleviated IS-induced neurobehavioral deficits and reduced brain infarct area and tissue damage. HSYA also significantly reduced hippocampal expression levels of LC3, HIF-1[Formula: see text], BNIP3, and Notch1. The beneficial effect of HSYA was generally superior to that of Edaravone. Molecular modeling suggested that HSYA may bind strongly to HIF-1[Formula: see text], BNIP3, and Notch1 but weakly to LC3. In conclusion, HSYA inhibits post-IS autophagy induction in the brain, possibly by suppressing HIF-1[Formula: see text], BNIP3 and Notch1. HSYA may have utility as a post-IS neuroprotective agent.

The Impact of Traditional Chinese Medicine on Mitophagy in Disease Models.

Mitophagy plays an important role in maintaining mitochondrial quality and cell homeostasis through the degradation of damaged, aged, and dysfunctional mitochondria and misfolded proteins. Many human diseases, particularly neurodegenerative diseases, are related to disorders of mitochondrial phagocytosis. Exploring the regulatory mechanisms of mitophagy is of great significance for revealing the molecular mechanisms underlying the related diseases. Herein, we summarize the major mechanisms of mitophagy, the relationship of mitophagy with human diseases, and the role of traditional Chinese medicine (TCM) in mitophagy. These discussions enhance our knowledge of mitophagy and its potential therapeutic targets using TCM.

Cyclovirobuxine D Induced-Mitophagy through the p65/BNIP3/LC3 Axis Potentiates Its Apoptosis-Inducing Effects in Lung Cancer Cells.

Mitophagy plays a pro-survival or pro-death role that is cellular-context- and stress-condition-dependent. In this study, we revealed that cyclovirobuxine D (CVB-D), a natural compound derived from Buxus microphylla, was able to provoke mitophagy in lung cancer cells. CVB-D-induced mitophagy potentiates apoptosis by promoting mitochondrial dysfunction. Mechanistically, CVB-D initiates mitophagy by enhancing the expression of the mitophagy receptor BNIP3 and strengthening its interaction with LC3 to provoke mitophagy. Our results further showed that p65, a transcriptional suppressor of BNIP3, is downregulated upon CVB-D treatment. The ectopic expression of p65 inhibits BNIP3 expression, while its knockdown significantly abolishes its transcriptional repression on BNIP3 upon CVB-D treatment. Importantly, nude mice bearing subcutaneous xenograft tumors presented retarded growth upon CVB-D treatment. Overall, we demonstrated that CVB-D treatment can provoke mitophagy and further revealed that the p65/BNIP3/LC3 axis is one potential mechanism involved in CVB-D-induced mitophagy in lung cancer cells, thus providing an effective antitumor therapeutic strategy for the treatment of lung cancer patients.

Akebia saponin D alleviates hepatic steatosis through BNip3 induced mitophagy.

Akebia Saponin D (ASD) is the most abundant constituent of the rhizome of Dipsacus asper Wall. The prior studies have shown that ASD alleviates hepatic steatosis targeted at the modulation of autophagy and exerts hepatoprotective effects through mitochondria. However, it is still unclear which signal transduction pathway that ASD increase autophagy and protect the mitochondria. The purpose of this paper was to explore the mechanisms through which ASD alleviates hepatic steatosis. ASD significantly reduced lipid accumulation in BRL cells. Furthermore, ASD significantly increased the mitophagy acting as increase the colocalization between mitochondria and punctate EGFP-LC3. ASD treatment increased the expression of BNip3, phospho-AMPK, prevented oleic acid (OA) induced LC3-II and phospho-mTOR expression. These effects were similar to the effects cotreatment with rapamycin. ASD treatment could not attenuate the expression of BNip3 blocked by chloroquine (CQ) or siRNA-mediated knockdown of BNip3. These results suggest that Akebia saponin D alleviates hepatic steatosis targeted at BNip3 mediated mitophagy. Activation of BNip3 via ASD may offer a new strategy for treating NAFLD.

Hyperoside protects against hypoxia/reoxygenation induced injury in cardiomyocytes by suppressing the Bnip3 expression.

AIMS: Role of hyperoside in protecting cardiomyocytes from ischemia/reperfusion induced injury has been proved. However, possible protecting mechanisms remain unclear. To fix the problem, an essential pro-apoptotic protein Bnip3 was studied in our experiments. METHODS AND RESULTS: Neonatal rat cardiomyocytes were used and submitted to hypoxia for 8h followed by reoxygenation for 2h to simulate the ischemia/reperfusion injury. Hypoxia/reoxygenation(H/R) induced damage to cardiomyocytes and the protective effect of hyperoside were examined by means of MTT assay. H/R-induced apoptosis was assessed by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL) and DNA Ladder assay. mRNA expression of Bnip3 was determined by use of quantitative real-time reverse transcription polymerase chain reaction assay. Protein levels of Bnip3, Bax, Bcl-2 and cleaved caspase-3 were examined using western-blot assay. Our results showed that H/R caused great damage to cardiomyocytes, upregulated the protein expressions of Bnip3, Bax, cleaved caspase3, and decreased the expression of the anti-apoptotic protein of Bcl-2. Whereas, compared with the H/R group, a decrease in activities of Bnip3, Bax, cleaved caspase3, and a promoting expression of Bcl-2 were detected in the H/R goup pretreated with hyperoside. CONCLUSION: It was concluded in our study that H/R-induced apoptotic effect in cardiomyocytes could be attenuated by hyperoside, and the protective role of hyperoside, if not completely, could be partly through the suppression of the pro-apoptotic gene Bnip3.

BNIP3 is essential for mitochondrial bioenergetics during adipocyte remodelling in mice.

AIMS/HYPOTHESIS: Adipose tissue is a highly versatile system in which mitochondria in adipocytes undergo significant changes during active tissue remodelling. BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) is a mitochondrial protein and a known mitochondrial quality regulator. In this study, we investigated the role of BNIP3 in adipocytes, specifically under conditions of peroxisome proliferator-activated receptor-γ (PPARγ)-induced adipose tissue remodelling. METHODS: The expression of BNIP3 was evaluated in 3T3-L1 adipocytes in vitro, C57BL/6 mice fed a high-fat diet and db/db mice in vivo. Mitochondrial bioenergetics was investigated in BNIP3-knockdown adipocytes after rosiglitazone treatment. A putative peroxisome proliferator hormone responsive element (PPRE) was characterised by promoter assay and electrophoretic mobility shift assay (EMSA). RESULTS: The protein BNIP3 was more abundant in brown adipose tissue than white adipose tissue. Furthermore, BNIP3 expression was upregulated by 3T3-L1 pre-adipocyte differentiation, starvation and rosiglitazone treatment. Conversely, BNIP3 expression in adipocytes decreased under various conditions associated with insulin resistance. This downregulation of BNIP3 was restored by rosiglitazone treatment. Knockdown of BNIP3 in adipocytes inhibited rosiglitazone-induced mitochondrial biogenesis and function, partially mediated by the 5' AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor γ, co-activator 1 α (PGC1α) signalling pathway. Rosiglitazone treatment increased the transcription level of Bnip3 in the reporter assay and the presence of the PPRE site in the Bnip3 promoter was demonstrated by EMSA. CONCLUSIONS/INTERPRETATION: The protein BNIP3 contributes to the improvement of mitochondrial bioenergetics that occurs on exposure to rosiglitazone. It may be a novel therapeutic target for restoring mitochondrial dysfunction under insulin-resistant conditions.