Collagen I protects human keratinocytes HaCaT against UVB injury via restoring PINK1/parkin-mediated mitophagy.

Collagen I is a major component of extracellular matrix in human skin, and is also widely used in a variety of skin-care products. In this study, we investigated the modulatory roles of collagen I on human immortalized keratinocytes HaCaT, especially when cells were irradiated with UVB. Interestingly, the cells grown on plates coated by molecular collagen I, but not fibrillar collagen I, acquired certain resistance against UVB damages, as shown by increased survival and reduced apoptosis. The accumulation of dysfunctional mitochondria in UVB-treated cells was attenuated by molecular collagen I-coating. Interestingly, molecular collagen I rescued the loss of mitochondrial biogenesis in cells treated with UVB. Loss of PINK1/parkin-mediated mitophagy was dominant for the accumulation of dysfunctional mitochondria after UVB irradiation. Of note, cells cultured on molecular collagen I-precoated plates exhibited reserved mitophagy after UVB irradiation, as reflected by the enhanced protein level of PINK1/parkin, increased mitochondrial ubiquitin and the co-localization of lysosomes and mitochondria. Moreover, in UVB-treated cells, inhibiting mitophagy by Cyclosporin A, or by silencing PINK1 or parkin, disturbed the resolution of mitochondrial stress and reduced the protective effect of molecular collagen I, indicating that mitophagy is pivotal for the protection of collagen I against UVB damage in keratinocytes HaCaT. Collectively, this study reveals an unexpected protective role of collagen I, which facilitates mitophagy to rescue cells under UVB irradiation, providing a new direction for clinical application of collagen products.

Lymphatic drainage patterns of malignant skin tumors in the head and neck region: a single-center retrospective study.

BACKGROUND: This study aimed to clarify the relationship between primary site and lymphatic drainage pattern for malignant skin tumors in the head and neck region. Malignant melanoma and squamous cell carcinoma in the head and neck region are known to have poor prognosis because of lymph node metastasis. Nevertheless, numerous aspects of lymphatic drainage patterns remain elusive. METHODS: We statistically analyzed data of 47 patients with malignant skin tumors in the head and neck region. Information was collected on the patients' clinical characteristics, primary tumor site, and lymphatic drainage patterns. RESULTS: The parotid lymph nodes drained the greatest amount of lymph from skin tumors of the head and neck. Important lymphatic drainage pathways were the superficial cervical nodes for primary tumors in the buccal/nasal region, level IA and level IB nodes for primary tumors in the lip region, the occipital nodes, posterior auricular nodes, and level VA nodes in the parietal/occipital region, and the preauricular nodes in the auricular region. CONCLUSION: These findings have considerable significance in terms of understanding lymphatic drainage patterns for malignant skin tumors in the head and neck and may be useful for clinical decision-making and when planning treatment. Further research and clinical applications are expected to contribute to an improved prognosis in patients with cutaneous head and neck malignancies.

Physiological and Functional Restoration of the Modiolus Using a Double Tendon Sling in Patients With Full-Thickness Oral Defects.

BACKGROUND: Reconstruction of the oral modiolus should be considered for facial defects involving the lip and cheek. We have performed free flap reconstruction using a novel technique that includes a vertical and a horizontal sling for full-thickness defects involving the oral commissure. PATIENTS AND METHODS: We used a free radial forearm flap and the palmaris longus tendon to correct lip and cheek defects in 6 patients (3 men, 3 women; age, 55-84 years). The tendon was divided into 2 portions for use as the double sling. One sling was used to connect the upper and lower stumps of the orbicularis oris in the vertical direction and the other to connect the vertical sling and masseter muscle. The outcomes assessed were esthetic appearance and functional results. RESULTS: The facial defects ranged in size from 3.0 cm × 4.0 cm to 7.0 cm × 6.0 cm and the mucosal defects from 1.5 cm × 4.0 cm to 6.5 cm × 5.0 cm. The length of the harvested tendon ranged from 11.5 to 13.5 cm. All flaps survived without complications. Good esthetic outcomes were achieved (excellent facial appearance, n = 1; no lip deviation or drooping, n = 5). Functionally, all patients had acceptable mouth opening and were able to eat a regular diet. CONCLUSIONS: Good physiological and functional results can be achieved using a double tendon sling for reconstruction of the modiolus in patients with full-thickness defects involving the oral commissure.

Increased mitochondrial fission induces NLRP3/cGAS-STING mediated pro-inflammatory pathways and apoptosis in UVB-irradiated immortalized human keratinocyte HaCaT cells.

Ultraviolet B (UVB) irradiation causes skin inflammation and apoptosis. Mitochondria are highly dynamic and undergo constant fusion and fission that are essential for maintaining physiological functions of cells. Although dysfunction of mitochondria has been implicated in skin damages, little is known about the roles of mitochondrial dynamics in these processes. UVB irradiation increases abnormal mitochondrial content but decreases mitochondrial volume in immortalized human keratinocyte HaCaT cells. UVB irradiation resulted in marked upregulation of mitochondrial fission protein dynamin-related protein 1 (DRP1) and downregulation of mitochondrial outer membrane fusion proteins 1 and 2 (MFN1 and MFN2) in HaCaT cells. Mitochondrial dynamics was discovered to be crucial for NLRP3 inflammasome and cGAS-STING pathway activation, as well as the induction of apoptosis. Inhibition of mitochondrial fission by treatments with a DRP1 inhibitor, mdivi-1, or with DRP1-targeted siRNA, efficiently prevented UVB-induced NLRP3/cGAS-STING mediated pro-inflammatory pathways or apoptosis in the HaCaT cells, whereas inhibition of mitochondrial fusion with MFN1and 2 siRNA increased these pro-inflammatory pathways or apoptosis. The enhanced mitochondrial fission and reduced fusion caused the up-regulation of reactive oxygen species (ROS). Application of an antioxidant, N-acetyl-l-cysteine (NAC), which scavenges excessive ROS, attenuated inflammatory responses through suppressing NLRP3 inflammasome and cGAS-STING pathway activation, and rescued cells from apoptosis caused by UVB-irradiation. Together, our findings revealed the regulation of NLRP3/cGAS-STING inflammatory pathways and apoptosis by mitochondrial fission/fusion dynamics in UVB-irradiated HaCaT cells, providing a new strategy for the therapy of UVB skin injury.

Impaired mitophagy causes mitochondrial DNA leakage and STING activation in ultraviolet B-irradiated human keratinocytes HaCaT.

Ultraviolet B (UVB) irradiation causes skin damages. In this study, we focus on the involvement of mitochondrial disorders in UVB injury. Surprisingly, UVB irradiation increases the amounts of mitochondria in human immortalized keratinocytes HaCaT. However, further analysis shows that ATP levels decreased by UVB treatment in accordance with the collapse of mitochondrial membrane potential (MMP), suggesting an accumulation of dysfunctional mitochondria in UVB-irradiated HaCaT cells. Mitophagy, mainly mediated by PINK1 and parkin, is critical for the elimination of damaged mitochondria. Western blot results show that the levels of both PINK1 and parkin are decreased in UVB-irradiated cells, indicating the impairment of mitophagy. Silencing the expression of PINK1 or parkin by transfection of siRNA shows essentially the same damage to the cells as UVB irradiation does, including increased mitochondrial amount, decreased MMP and ATP production, and enhanced apoptosis, evidencing that repression of PINK1/parkin-mediated mitophagy plays a primary cause of UVB-caused cells damages. We previously found that HaCaT cells exposed to UVB showed activation of the cGAS-STING pathway and apoptosis. Here, silencing PINK1 or parkin also increases the protein levels of cGAS and STING, facilitates nuclear accumulation of NF-κB, and promotes the transcription of IFNß, suggesting for the activation of STING pathway. Mitophagy impairment either by UVB-irradiation or by PINK1/parkin silencing initiates caspase-3-mediated apoptosis, as shown by the activation of caspase-3 and cleavage of PARP, as well as the increase of Hoechst-positive stained cells and Annexin V-positive cells. Further studies find that Bax-mediated permeabilization of mitochondrial membrane is critical for cell apoptosis, as well as the cytosolic leakage of mtDNA in UVB-treated cells, which results in cGAS-STING activation, and these processes are negatively-regulated by PINK1/parkin-mediated mitophagy. This study reveals the involvement of dysfunctional mitochondria due to impaired mitophagy in the damaging effect of UVB irradiation on HaCaT cells. Restoring the mitophagy has the potential to be developed as a new strategy to protect skin from UVB damages.

Silibinin ameliorates STING-mediated neuroinflammation via downregulation of ferroptotic damage in a sporadic Alzheimer's disease model.

Ferroptosis, an iron-dependent cell death, is caused by lipid peroxidation. Noteworthily, accumulation of iron and lipid peroxidation are found in the proximity of the neuritic plaque, a hallmark of Alzheimer's disease (AD), but the relationship between ferroptosis and neuroinflammation in AD is unclear. Silibinin, extracted from the Silybum marianum, is possibly developed as an agent for AD treatment from its neuroprotective effect, but the effect of silibinin on sporadic AD that accounts for more than 95% of AD remains unclear. To determine whether silibinin alleviates the pathogenesis of sporadic AD and investigate the underlying mechanisms, STZ-treated HT22 murine hippocampal neurons and intracerebroventricular injection of streptozotocin (ICV-STZ) rats, a sporadic AD model, were used in this study. Results show that silibinin not only promotes survival of STZ-treated HT22 cells, but also ameliorates the cognitive impairment and anxiety/depression-like behavior of ICV-STZ rats. We here demonstrate that silibinin evidently inhibits the protein level of p53 as well as upregulates the protein level of cystine/glutamate antiporter SLC7A11 and ferroptosis inhibitor GPX4, but not p21, leading to the protection against STZ-induced ferroptotic damage. Immunofluorescent staining also shows that accumulation of lipid peroxidation induced by ferroptotic damage leads to increased fluorescence of 8-oxo-deoxyguanosine (8-OHDG), a maker of oxidized DNA. The oxidized DNA then leaks to the cytoplasm and upregulates the expression of the stimulator of interferon gene (STING), which triggers the production of IFN-ß and other inflammatory cascades including NF-κB/TNFα and NLRP3/caspase 1/IL-1ß. However, the treatment with silibinin blocks the above pathological changes. Moreover, in HT22 cells with/without STZ treatment, GPX4-knockdown increases the protein level of STING, indicating that the ferroptotic damage leads to the activation of STING signaling pathway. These results imply that silibinin exerts neuroprotective effect on an STZ-induced sporadic AD model by downregulating ferroptotic damage and thus the downstream STING-mediated neuroinflammation.

Silibinin alleviates ferroptosis of rat islet ß cell INS-1 induced by the treatment with palmitic acid and high glucose through enhancing PINK1/parkin-mediated mitophagy.

Type 2 diabetes (T2DM) is induced by the abundance of glucose and lipids, which causes glucolipotoxicity to the pancreatic ß-cells. Silibinin is a natural flavonoid possessing the regulatory activity on insulin production and therapeutic activity in diabetic mice; however, its effect on glucolipotoxicity is not fully explained. This in vitro study investigates the effects of silibinin on palmitic acid (PA) and high glucose (HG)-induced cell loss and ferroptosis of rat insulinoma INS-1 cells. In the cells treated with PA and HG, expressions of glucose transporter 4 (Glut4) and carnitine acyltransferase I (CPT1) for ß-oxidation of fatty acids are reduced. Mitochondria are the metabolic organelles for glucose and fatty acids. The mitochondrial membrane potential (MMP) and ATP production were decreased, while the ROS level was elevated in the cells treated with PA and HG, indicating an induction of mitochondrial disorder. Cell loss was partially rescued by ferroptosis inhibition, suggesting an involvement of ferroptosis in the cells treated with PA and HG. More importantly, the increases in total iron, lipid ROS, MDA and COX-2, and the decrease in ferroptosis inhibitory molecules GSH, GPX4 and FSP1 appeared in the cells treated with PA and HG, confirming the occurrence of ferroptosis. Moreover, PINK1/parkin-mediated mitophagy, a vital process for selective elimination of damaged mitochondria, was blocked. Interestingly, silibinin rescued the mitochondria, restricted the ferroptosis and restored the mitophagy. By using the pharmacological stimulator and inhibitor of mitophagy, and si-RNA transfection to silence PINK1 expression, silibinin's protective effect against ferroptosis caused by PA and HG treatment was found to depend on mitophagy. Collectively, our current study reveals the new mechanisms for the protection of silibinin against the injury of INS-1 cells treated with PA and HG, elucidates the participation of ferroptosis in glucolipotoxicity, highlighting the involvement of mitophagy in defense against ferroptotic cell death.

Restoration of lymph flow by flap transfer can prevent severe lower extremity lymphedema after inguino-pelvic lymphadenectomy.

PURPOSE: Severe lymphedema is difficult to treat because of the associated extensive scar formation. Therefore, preventing scar formation might alleviate the severity of lymphedema following lymphadenectomy. In this study, we evaluated the usefulness of flap transfer, performed immediately after lymphadenectomy, for preventing scar formation. METHODS: Twenty-three patients with subcutaneous malignancy in a lower extremity, who underwent inguino-pelvic lymphadenectomy, were divided into groups based on whether flap transfer was performed. The severity of lymphedema was categorized according to the ratio of the circumference of the affected extremity to that of the unaffected extremity, as mild (< 20% increase in volume), moderate (20-40%), or severe (> 40%). RESULTS: In the 18 patients who underwent lymphadenectomy without flap transfer, lymphedema was classified as mild in 7, moderate in 7, and severe in 4. In the five patients who underwent lymphadenectomy with flap transfer, lymphedema was classified as mild in 4 and moderate in 1. This difference between the groups did not reach significance. CONCLUSIONS: The findings of this study suggest that flap transfer may help prevent scar formation and contribute to the restoration of lymph flow after lymphadenectomy.

Long-term prognosis of 452 moyamoya disease patients with and without revascularization under perfusion-based indications.

OBJECTIVES: To evaluate the long-term outcomes of patients treated under our perfusion-based strategy and assess whether conservative treatment without surgical treatment under our strategy is acceptable. MATERIALS AND METHODS: A total of 315 adult and 137 pediatric MMD patients (follow-up period ≥ 3 years from 2001 to 2020) were included. Follow-up events in each patient group (pediatric or adult, surgically treated or conservatively treated) were evaluated and compared to each other using a log-rank test. Risk factors for stroke and nonstroke events were also investigated using a multivariate Cox proportional hazard model. RESULTS: In adult-onset patients, the stroke event rates (person-year %) were not different between surgically treated patients and conservatively treated patients (2.00 % vs. 1.59 %, p = 0.558); however, conservative patients showed a higher stroke rate than surgically treated hemispheres (0.34 %; p = 0.025) and hemorrhagic stroke was the major type (18/26, 69.2 %). Hemorrhagic onset was associated with increased risk of stroke in adults (hazard ratio (95 % confidence interval) = 2.43 (1.10-5.36)). In pediatric-onset patients, no conservatively treated patients experienced stroke; however, nonstroke events occurred more frequently than in surgically treated hemispheres (4.86 % vs. 1.71 %, p = 0.020 for transient ischemic attack; and 7.91 % vs. 1.31 %, p < 0.001 for asymptomatic progression on magnetic resonance angiography). CONCLUSIONS: In adult patients, conservatively treated patients experienced stroke more frequently, especially hemorrhagic stroke. An additive strategy to prevent stroke in hemorrhagic-onset patients without hemodynamic disturbance seems to be needed. Pediatric patients with mild hemodynamic disturbance can be safely observed without initial surgical intervention, but close follow-up for disease progression is necessary.

IGF-1R/YAP signaling pathway is involved in collagen V-induced insulin biosynthesis and secretion in rat islet INS-1 cells.

PURPOSE: Type V collagen (collagen V) is one of the important components of extracellular matrix (ECM) in pancreas. We previously reported that pre-coating collagen V on the culture dishes enhanced insulin production in INS-1 rat pancreatic ß cells. In this study, we investigate the underlying mechanism. RESULTS: Insulin biosynthesis and secretion are both increased in INS-1 cells cultured on collagen V-coated dishes, accompanied by the reduced nuclear translocation of Yes-associated protein (YAP), a transcriptional co-activator. YAP, the downstream effector of Hippo signaling pathway, plays an important role in the development and function of pancreas. Inhibition of YAP activation by verteporfin further up-regulates insulin biosynthesis and secretion. Silencing large tumor suppressor (LATS), a core component of Hippo pathway which inhibits activity of YAP by phosphorylation, by siRNA transfection inhibits both insulin biosynthesis and secretion. In the present study, the protein level of insulin-like growth factor 1 receptor (IGF-1 R), detected as the upstream molecule of YAP, is reduced in the INS-1 cells cultured on the dishes coated with collagen V. The silencing of IGF-1 R by siRNA transfection further enhances insulin biosynthesis and secretion. IGF-1 treatment reduces collagen V-induced up-regulation of insulin biosynthesis and secretion, accompanying the increased nuclear YAP. CONCLUSION: Inhibition of IGF-1 R/YAP signal pathway is involved in collagen V-induced insulin biosynthesis and secretion in INS-1 cells.

Silibinin relieves UVB-induced apoptosis of human skin cells by inhibiting the YAP-p73 pathway.

Excessive exposure to UVB induces skin diseases. Silibinin, a flavonolignan used for treating liver diseases, is found to be effective against UVB-caused skin epidermal and dermal cell damage. In this study we investigated the molecular mechanisms underlying. Human nonmalignant immortalized keratinocyte HaCaT cells and neonatal human foreskin fibroblasts HFFs were exposed to UVB irradiation. We showed that pre-treatment with silibinin dose-dependently decreased UVB-induced apoptosis of HaCaT cells. Furthermore, we showed that silibinin treatment inhibited nuclear translocation of YAP after UVB irradiation. Molecular docking analysis and DARTS assay confirmed the direct interaction of silibinin with YAP. Silencing YAP by siRNA had no influence on the survival of HaCaT cells, whereas inhibiting classical YAP-TEAD signaling pathway by siRNA targeting TEAD1 or its pharmaceutical inhibitor verteporfin further augmented UVB-induced apoptosis, suggesting that YAP-TEAD pathway was prosurvival, which did not participate in the protective effect of silibinin. We then explored the pro-apoptotic YAP-p73 pathway. p73 was upregulated in UVB-irradiated cells, but reduced by silibinin cotreatment. The mRNA and protein levels of p73 target genes (PML, p21 and Bax) were all increased by UVB but decreased by silibinin co-treatment. Inhibiting p73 by using siRNA reduced UVB-induced apoptosis, suggesting that downregulation of p73 was responsible for the cytoprotective effect of silibinin. In HFFs, the upregulated YAP-p73 pathway by UVB irradiation was also suppressed by silibinin. Collectively, YAP-p73 pathway is a major cause of the death of UVB-exposed epidermal HaCaT cells and dermal HFFs. Silibinin directly inhibits YAP-p73 pathway, exerting the protective action on UVB-irradiated skin cells.

Oral Administration of Silibinin Ameliorates Cognitive Deficits of Parkinson's Disease Mouse Model by Restoring Mitochondrial Disorders in Hippocampus.

Besides motor disorder, cognitive dysfunction is also common in Parkinson's disease (PD). Essentially no causal therapy for cognitive dysfunction of PD exists at present. In this study, a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD was used to analyze the neuroprotective potential of orally administered silibinin, a proverbial hepatoprotective flavonoid derived from the herb milk thistle (Silybum marianum). Results demonstrated that silibinin administration significantly attenuated MPTP-induced cognitive impairment in behavioral tests. Nissl staining results showed that MPTP injection significantly increases the loss of neurons in the hippocampus. However, these mice were protected by oral administration of silibinin, accompanying reduction in the cell apoptosis in the hippocampus. The hippocampal aggregates of α-synuclein (α-syn) appeared in MPTP-injected mice, but were significantly decreased by silibinin treatment. MPTP injection induced oxidative stress, as evidenced by increased malondialdehyde (MDA) and decreased superoxide dismutase (SOD). The oxidative stress was alleviated by silibinin treatment. Mitochondrial disorder including the decline of mitochondrial membrane potential (MMP) was another signature in the hippocampus of MPTP-treated mice, accompanying increased mitochondrial fission and decreased fusion. Silibinin administration restored these mitochondrial disorders, as expected for the protection against MPTP injury. These findings suggest that silibinin has a potential to be further developed as a therapeutic candidate for cognitive dysfunction in PD.

Collagens I and V differently regulate the proliferation and adhesion of rat islet INS-1 cells through the integrin ß1/E-cadherin/ß-catenin pathway.

Extracellular matrix (ECM) plays an important role in tissue repair, cell proliferation, and differentiation. Our previous study showed that collagen I and collagen V differently regulate the proliferation of rat pancreatic ß cells (INS-1 cells) through opposite influences on the nuclear translocation of ß-catenin. In this study, we investigated the ß-catenin pathway in INS-1 cells on dishes coated with collagen I or V. We found that nuclear translocation of the transcription factor Yes-associated protein (YAP) was enhanced by collagen I and suppressed by collagen V, but had no effect on INS-1 cell proliferation. Morphologically, INS-1 cells on collagen V-coated dishes showed stronger cell-to-cell adhesion, while the cells on collagen I-coated dishes showed weaker cell-to-cell adhesion in comparison with the cells on non-coated dishes. E-cadherin played an inhibitory role in the proliferation of INS-1 cells cultured on collagen I or collagen V coated dishes via regulation of the nuclear translocation of ß-catenin. Integrin ß1 was enhanced with collagen I, while it was repressed with collagen V. The integrin ß1 pathway positively regulated the cell proliferation. Inhibition of integrin ß1 pathway restored the protein level of E-cadherin and inhibited the nuclear translocation of ß-catenin in the cells on collagen I-coated dishes, but no effect was observed in the cells on collagen V-coated dishes. In conclusion, collagen I enhances the proliferation of INS-1 cells via the integrin ß1 and E-cadherin/ß-catenin signaling pathway. In INS-1 cells on collagen V-coated dishes, both integrin ß1 and E-cadherin/ß-catenin signal pathways are involved in the inhibition of proliferation.

Toyo virus, a novel member of the Kaisodi group in the genus Uukuvirus (family Phenuiviridae) found in Haemaphysalis formosensis ticks in Japan.

Ticks are important vector arthropods that transmit various pathogens to humans and other animals. Tick-borne viruses are of particular concern to public health as these are major agents of emerging and re-emerging infectious diseases. The Phenuiviridae family of tick-borne viruses is one of the most diverse groups and includes important human pathogenic viruses such as severe fever with thrombocytopenia syndrome virus. Phenuivirus-like sequences were detected during the surveillance of tick-borne viruses using RNA virome analysis from a pooled sample of Haemaphysalis formosensis ticks collected in Ehime, Japan. RT-PCR amplification and Sanger sequencing revealed the nearly complete viral genome sequence of all three segments. Comparisons of the viral amino acid sequences among phenuiviruses indicated that the detected virus shared 46%-70% sequence identity with known members of the Kaisodi group in the genus Uukuvirus. Furthermore, phylogenetic analysis of the viral proteins showed that the virus formed a cluster with the Kaisodi group viruses, suggesting that this was a novel virus, which was designated "Toyo virus" (TOYOV). Further investigation of TOYOV is needed, and it will contribute to understanding the natural history and the etiological importance of the Kaisodi group viruses.

Combined internal and external negative pressure wound therapy: breakthrough treatment for lymphocutaneous intractable fistula.

PURPOSE: Lymphocutaneous fistula after lymph node dissection is intractable, yet there is no established treatment strategy. This study demonstrates the wound closure time achieved by a new method of combined internal and external negative pressure wound therapy (CIEN) in patients with lymphocutaneous fistula. METHODS: The subjects of this study were six consecutive patients with lymphocutaneous fistula after lymphatic surgery, who were treated with CIEN between 2018 and 2020. The CIEN technique can be summarized as follows: first, internal foam is inserted into the fistula from the opening of the fenestration. Next, a slightly larger area of external foam is applied above the fistula flap outside the external margin of the foam-filled fistula. After bridging the internal foam and external foam, negative-pressure wound therapy is carried out on this bridging foam block. RESULTS: CIEN led to rapid and complete wound healing in all six patients. Fistula flap margin ischemia developed in one patient, but adjusting the mode and pressure settings resulted in improvement. Three patients suffered contact dermatitis. There were no signs of tumor or fistula recurrence in any patients after at least 3 months of follow-up. CONCLUSION: CIEN is an effective and less invasive treatment modality than the conventional method of managing lymphocutaneous fistula.

Type I collagen inhibits adipogenic differentiation via YAP activation in vitro.

Extracellular matrix (ECM) has a marked influence on adipose tissue development. Adipose tissue formation is initiated with proliferation of preadipocytes and migration before undergoing further differentiation into mature adipocytes. Previous studies showed that collagen I (col I) provides a good substratum for 3T3-L1 preadipocytes to grow and migrate. However, it remains unclear whether and how col I regulates adipogenic differentiation of preadipocytes. This study reports that lipid accumulation, representing in vitro adipogenesis of the 3T3-L1 preadipocytes or the mouse primary adipocyte precursor cells derived from subcutaneous adipose tissue in the inguinal region is inhibited by the culture on col I, owing to downregulation of adipogenic factors. Previous study shows that col I enhances 3T3-L1 cell migration via stimulating the nuclear translocation of yes-associated protein (YAP). In this study, we report that downregulation of YAP is associated with in vitro adipogenesis of preadipocytes as well as with in vivo adipose tissue of high-fat diet fed mice. Increased expression of YAP in the cells cultured on col I-coated dishes is correlated with repression of adipogenic differentiation processes. The inactivation of YAP using YAP inhibitor, verteporfin, or YAP small-interfering RNA enhanced adipogenic differentiation and reversed the inhibitory effect of col I. Activation of YAP either by the transfection of YAP plasmid or the silence of large tumor suppressor 1 (LATS1), an inhibitory kinase of YAP, inhibited adipogenic differentiation. The results indicate that col I inhibits adipogenic differentiation via YAP activation in vitro.

Estrogen receptors participate in silibinin-caused nuclear translocation of apoptosis-inducing factor in human breast cancer MCF-7 cells.

Our previous studies showed that silibinin promoted activation of caspases to induce apoptosis in human breast cancer MCF-7 cells by down-regulating the protein expression level of estrogen receptor (ER) α and up-regulating ERß. Recently, it has been reported that silibinin-induced apoptosis also involved nuclear translocation of apoptosis-inducing factor (AIF). Here we report that silibinin induces nuclear translocation of AIF through the down-regulation of ERα and up-regulation of ERß in a concentration dependent manner in MCF-7 cells. AIF knockdown with siRNA significantly reverses silibinin-induced apoptosis. The nuclear translocation of AIF is enhanced by treatment with MPP, an ERα antagonist, and blocked with PPT, an ERα agonist. In contrast to ERα activity, the nuclear AIF is increased with an ERß agonist, DPN and blocked with an ERß antagonist, PHTPP. Autophagy, negatively regulated by ERα, positively controls AIF-mediated apoptosis, as evidenced by the preventive effect of autophagy inhibitor 3-MA and siRNA targeting LC3, on the nuclear translocation of AIF and cell death induced by silibinin co-treatment with or without MPP. In sum we conclude that AIF in nuclei is involved in silibinin-induced apoptosis, and the nuclear translocation of AIF is increased by down-regulated ERα pathway and/or up-regulated ERß pathway in MCF-7 cells, accompanying up-regulation of autophagy.

Silibinin-induced mitochondria fission leads to mitophagy, which attenuates silibinin-induced apoptosis in MCF-7 and MDA-MB-231 cells.

We reported previously that higher doses (150-250 µM) of silibinin enhanced fission and inhibited fusion of mitochondria, accompanying apoptosis of double-positive breast cancer cell line MCF-7 cells and triple-negative breast cancer cell line MDA-MB-231 cells. We report here three important questions yet unclarified in the previous study; 1) Whether enhanced fission of mitochondria by the treatment of silibinin leads to mitophagy, 2) Whether mitophagy positively contributes to apoptosis and 3) Whether estrogen receptor-positive (ER+) MCF-7 cells and estrogen receptor-negative (ER-) MDA-MB-231 cells are affected in a different way by silibinin treatment, since silibinin often works through ERs signaling pathway. Mitophagy driven by Pink1/Parkin signaling, plays an important role in eliminating damaged mitochondria. Indeed, increased expression of Pink1 and the recruitment of Parkin and LC3-II to mitochondria by the treatment with silibinin account for silibinin induction of mitophagy. In this study, the effects of mitochondrial division inhibitor 1 (mdivi-1) and small interfering RNA targeting dynamin-related protein 1 (DRP1) were examined to reveal the effect of mitochondrial fission on mitophagy. As expected, mdivi-1 or siRNA targeting DRP1 reversed silibinin-induced mitochondrial fission due to down-regulation in the expression of DRP1. Inhibition of mitochondrial fission by mdivi-1 prevented induction of mitophagy as well as autophagy in both MCF-7 and MDA-MB-231 cells, indicating that silibinin-induced mitochondrial fission leads to mitophagy. Inhibition of mitochondrial fission efficiently prevented silibinin-induced apoptosis in MCF-7 and MDA-MB-231 cells in our previous work, and the second point of the present study, inhibition of mitophagy by Pink1 or Parkin knockdown increased silibinin-induced apoptosis of these cells, respectively, suggesting that the mitophagy induced by silibinin treatment serves as a cytoprotective effect, resulting in reduction of apoptosis of cancer cells in both cells. In the third point, we studied whether estrogen receptors (ERs) played a role in silibinin-induced mitophagy and apoptosis in MCF-7 and MDA-MB-231 cells. ERα and ERß are not involved in silibinin-induced mitophagic process in MCF-7 and MDA-MB-231 cells. These findings demonstrated that silibinin induced mitochondria fission leads to mitophagy, which attenuates silibinin-induced apoptosis not through ERs-Pink1 or -Parkin pathway in MCF-7 and MDA-MB-231.

Natural flavonoid silibinin promotes the migration and myogenic differentiation of murine C2C12 myoblasts via modulation of ROS generation and down-regulation of estrogen receptor α expression.

Skeletal muscle regeneration is a complex process, involving the proliferation, migration, and differentiation of myoblasts. Recent studies suggest that some natural flavanones stimulate myogenesis. However, the effect of plant estrogen, silibinin, on the regulation of myoblast behaviors is unclarified. In this study, we investigated the effects of silibinin on immortalized murine myoblast C2C12 in the aspects of proliferation, migration, differentiation along with underlying mechanisms. The results show that silibinin at concentrations below 50 µM enhanced the migration and differentiation of C2C12 cells, but had no effect on cell proliferation. Silibinin significantly promoted the production of ROS, which appeared to play important roles in the migration and differentiation of the myoblasts. Interestingly, among ROS, the superoxide anion and hydroxyl radical were associated with the migration, whereas hydrogen peroxide contributed to the myogenic differentiation. We used ER agonist and antagonist to explore whether estrogen receptors (ERs), which are affected by silibinin treatment in the silibinin-enhanced C2C12 migration and differentiation. Migration was independent of ERs, whereas the differentiation was associated with decreased ERα activity. In summary, silibinin treatment increases ROS levels, leading to the promotion of migration and myogenic differentiation. Negative regulation ERα of differentiation but not of migration may suggest that ERα represses hydrogen peroxide generation. The effect of silibinin on myoblast migration and differentiation suggests that silibinin may have therapeutic benefits for muscle regeneration.

Silibinin inhibits migration and invasion of breast cancer MDA-MB-231 cells through induction of mitochondrial fusion.

Human triple negative breast cancer cells, MDA-MB-231, show typical epithelial to mesenchymal transition associated with cancer progression. Mitochondria play a major role in cancer progression, including metastasis. Changes in mitochondrial architecture affect cellular migration, autophagy and apoptosis. Silibinin is reported to have anti-breast cancer effect. We here report that silibinin at lower concentrations (30-90 µM) inhibits epithelial to mesenchymal transition (EMT) of MDA-MB-231, by increasing the expression of epithelial marker, E-cadherin, and decreasing the expression of mesenchymal markers, N-cadherin and vimentin. Besides, silibinin inhibition of cell migration is associated with reduction in the protein expression of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) and paxillin. In addition, silibinin treatment increases mitochondrial fusion through down-regulating the expression of mitochondrial fission-associated protein dynamin-related protein 1 (DRP1) and up-regulating the expression of mitochondrial fusion-associated proteins, optic atrophy 1, mitofusin 1 and mitofusin 2. Silibinin perturbed mitochondrial biogenesis via down-regulating the levels of mitochondrial biogenesis regulators including mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator (PGC1) and nuclear respiratory factor (NRF2). Moreover, DRP1 knockdown or silibinin inhibited cell migration, and MFN1&2 knockdown restored it. Mitochondrial fusion contributes to silibinin's negative effect on cell migration. Silibinin decreased reactive oxygen species (ROS) generation, leading to inhibition of the NLRP3 inflammasome activation. In addition, knockdown of mitofusin 1&2 (MFN 1&2) relieved silibinin-induced inhibition of NLRP3 inflammasome activation. Repression of ROS contributes to the inhibition of the expression of NLRP3, caspase-1 and IL-ß proteins as well as of cell migration. Taken together, our study provides evidence that silibinin impairs mitochondrial dynamics and biogenesis, resulting in reduced migration and invasion of the MDA-MB-231 breast cancer cells.