Aerosol inhalation of total ginsenosides repairs acute lung injury and inhibits pulmonary fibrosis through SMAD2 signaling-mediated mechanism.

BACKGROUND: Pulmonary fibrosis (PF) is a progressive lung disease caused by previous acute lung injury (ALI), but there is currently no satisfactory therapy available. Aerosol inhalation of medicine is an effective way for treating PF. Total ginsenosides (TG) shows potential for the treatment of ALI and PF, but the effects of inhaled TG remain unclear. PURPOSE: To determine the therapeutic effects of TG in ALI and PF, to assess the superiority of the inhaled form of TG over the routine form, and to clarify the mechanism of action of inhaled TG. METHODS: Ultrahigh-performance liquid chromatography coupled with Q-Exactive Orbitrap mass spectrometry (UPLC-QE-MS) was applied to determine the chemoprofile of TG. A mouse model of ALI and PF was established to evaluate the effects of inhaled TG by using bronchoalveolar lavage fluid (BALF) analysis, histopathological observation, hydroxyproline assay, and immunohistochemical analysis. Primary mouse lung fibroblasts (MLF) and human lung fibroblast cell line (HFL1) were applied to determine the in vitro effects and mechanism of TG by using cell viability assay, quantitative real time PCR (qPCR) assay, and western blot (WB) analysis. RESULTS: The UPLC-QE-MS results revealed the main types of ginsenosides in TG, including Re (14.15 ± 0.42%), Rd (8.42 ± 0.49%), Rg1 (6.22 ± 0.42%), Rb3 (3.28 ± 0.01%), Rb2 (3.09 ± 0.00%), Rc (2.33 ± 0.01%), Rg2 (2.09 ± 0.04%), Rb1 (1.43 ± 0.24%), and Rf (0.13 ± 0.06%). Inhaled TG, at dosages of 10, 20, and 30 mg/kg significantly alleviated both ALI and PF in mice. Analyses of BALF and HE staining revealed that TG modulated the levels of IFN-γ, IL-1ß, and TGF-ß1, reduced inflammatory cell infiltration, and restored the alveolar architecture of the lung tissues. Furthermore, HE and Masson's trichrome staining demonstrated that TG markedly decreased fibroblastic foci and collagen fiber deposition, evidenced by the reduction of blue-stained collagen fibers. Hydroxyproline assay and immunohistochemical analyses indicated that TG significantly decreased hydroxyproline level and down-regulated the expression of Col1a1, Col3a1, and α-sma. The inhaled administration of TG demonstrated enhanced efficacy over the oral route when comparable doses were used. Additionally, inhaled TG showed superior safety and therapeutic profiles compared to pirfenidone, as evidenced by a CCK8 assay, which confirmed that TG concentrations ranging from 20 to 120 µg/ml were non-cytotoxic. qPCR and WB analyses revealed that TG, at concentrations of 25, 50, and 100 µg/ml, significantly suppressed the phosphorylation of smad2 induced by TGF-ß1 and down-regulated the expression of fibrotic genes and proteins, including α-sma, Col1a1, Col3a1, and FN1, suggesting an anti-fibrotic mechanism mediated by the smad2 signaling pathway. In vitro, TG's safety and efficacy were also found to be superior to those of pirfenidone. CONCLUSIONS: This study demonstrates, for the first time, the therapeutic efficacy of inhaled TG in treating ALI and PF. Inhaled TG effectively inhibits inflammation and reduces collagen deposition, with a particular emphasis on its role in modulating the Smad2 signaling pathway, which is implicated in the anti-fibrotic mechanism of TG. The study also highlights the superiority of inhaled TG over the oral route and its favorable safety profile in comparison to pirfenidone, positioning it as an ideal alternative for ALI and PF therapy.

PBMC-mediated modulation of macrophage polarization in RAW264.7 cells through STAT1/STAT6 signaling cascades.

Peripheral blood mononuclear cells (PBMC), sourced autologously, offer numerous advantages when procured: easier acquisition process, no in vitro amplification needed, decreased intervention and overall increased acceptability make PBMC an attractive candidate for cell therapy treatment. However, the exact mechanism by which PBMC treat diseases remains poorly understood. Immune imbalance is the pathological basis of many diseases, with macrophages playing a crucial role in this process. However, research on the role and mechanisms of PBMC in regulating macrophages remains scarce. This study employed an in vitro co-culture model of PBMC and RAW264.7 macrophages to explore the role and mechanisms of PBMC in regulating macrophages. The results showed that the co-culturing led to decreased expression of inflammatory cytokines and increased expression of anti-inflammatory cytokines in RAW264.7 or in the culture supernatant. Additionally, the pro-inflammatory, tissue matrix-degrading M1 macrophages decreased, while the anti-inflammatory, matrix-synthesizing, regenerative M2 macrophages increased in both RAW264.7 and monocytes within PBMC. Moreover, co-cultured macrophages exhibited a significantly decreased p-STAT1/STAT1 ratio, while the p-STAT6/STAT6 ratio significantly increased. This suggests that PBMC may inhibit M1 macrophage polarization by blocking STAT1 signaling cascades and may promote M2 macrophage polarization through the activation of STAT6 signaling cascades. Overall, this study sheds light on the role and mechanism of PBMC in regulating macrophages. Moreover, it was found that monocytes within co-cultured PBMC differentiated into M2 macrophages in the presence of macrophages. This finding provides experimental evidence for the use of PBMC in treating inflammatory diseases, especially macrophage-depleting inflammatory diseases such as osteoarthritis.

Corrigendum: Nanofat lysate ameliorates pain and cartilage degradation of osteoarthritis through activation of TGF-ß-Smad2/3 signaling of chondrocytes.

[This corrects the article DOI: 10.3389/fphar.2023.900205.].

Cytotoxicity of dioncophylline A and related naphthylisoquinolines in leukemia cells, mediated by NF-κB inhibition, angiogenesis suppression, G2/M cell cycle arrest, and autophagy induction.

BACKGROUND: Inhibition of NF-κB activity represents a strategy to treat acute myeloid leukemia, one of the most lethal leukemia types. Naphthylisoquinolines (NIQs) are cytotoxic alkaloids from lianas of the families Ancistrocladaceae and Dioncophyllaceae, which are indigenous to tropical rainforests. PURPOSE: Uncovering therapeutic possibilities and underlying molecular mechanisms of dioncophylline A and its derivatives towards NF-κB related cellular processes. METHODS: Resazurin-based cell viability assay was performed for dioncophylline A and three derivatives on wild-type CCRF-CEM and multidrug-resistant CEM/ADR5000 cells. Transcriptome analysis was executed to discover cellular functions and molecular networks associated with dioncophylline A treatment. Expression changes obtained by mRNA microarray hybridization were confirmed using qRT-PCR. Molecular docking was applied to predict the affinity of the NIQs with NF-κB. To validate the in silico approach, NF-κB reporter assays were conducted on HEK-Blue™ Null1 cells. Cell death mechanisms and cell cycle arrest were studied using flow cytometry. The potential activity on angiogenesis was evaluated with the endothelial cell tube formation assay on HUVECs using fluorescence microscopy. Intracellular NF-κB location in HEK-Blue™ Null1 cells was visualized with immunofluorescence. Finally, the anti-tumor activity of dioncophylline A was studied by a xenograft zebrafish model in vivo. RESULTS: Our study demonstrated that dioncophylline A and its derivatives exerted potent cytotoxicity on leukemia cells. Using Ingenuity Pathway Analysis, we identified the NF-κB network as the top network, and docking experiments predicted dioncophylline A and two of its derivatives sharing the same binding pocket with the positive control compound, triptolide. Dioncophylline A showed the best inhibitory activity in NF-κB reporter assays compared to its derivatives, caused autophagy rather than apoptosis, and induced G2/M arrest. It also prevented NF-κB translocation from the cytoplasm to the nucleus. Tube formation as an angiogenesis marker was significantly suppressed by dioncophylline A treatment. Finally, the remarkable anti-tumor activity of dioncophylline A was proven in zebrafish in vivo. CONCLUSION: Taken together, we report for the first time the molecular mechanism behind the cytotoxic effect of dioncophylline A on leukemia cells. Dioncophylline A showed strong cytotoxic activity, inhibited NF-κB translocation, significantly affected the NF-κB in silico and in vitro, subdued tube formation, induced autophagy, and exerted antitumor activity in vivo. Our findings enlighten both the cellular functions including the NF-κB signaling pathway and the cytotoxic mechanism affected by dioncophylline A.

Corn stigma ameliorates hyperglycemia in zebrafish and GK rats of type 2 diabetes.

ETHNOPHARMACOLOGICAL RELEVANCE: Cornstigma (CS), derived from the stigma and style of gramineous plant Zeamays. The medicinal use of CS can be traced back to DianNanMateriaMedica. LingnanMedicinalPlantsCompendium records its effectiveness in ameliorating diabetes. Diabetes is a metabolic disorder characterized by hyperglycemia and the consequent chronic complications of kidney, heart, brain and other organs, which pose a significant threat to human health. CS has shown great potential in relieving hyperglycemia associated with diabetes. However, the mechanism of CS in treating diabetes remains unclear. AIM OF THE STUDY: To explore the pathogenesis of diabetes and the mechanism of CS improving hyperglycemia in diabetes. MATERIALS AND METHODS: We measured apigenin and luteolin contents in CS by UPLC/MS/MS method. Selecting Wistar rats as normal group, and GK rats as model group. For rats, we detected glucose and lipid metabolism indicators, including GHb, AST, ALT, U-Glu, UA, U-TP, U-ALB, and ACR after treatment. For zebrafish, we utilized alloxan and sucrose to establish the diabetes model. Measuring zebrafish blood glucose is employed to evaluate the hypoglycemic capability of CS. In order to explore the mechanism of CS in treating diabetes, we sequenced the transcriptome of zebrafish, compared differentially expressed genes of normal, diabetic, and CS-treated group, and validated multiple enrichment pathways by PCR. RESULTS: CS can improve blood glucose levels in both GK rats and diabetic zebrafish. For rats, CS partially restored glucose and lipid metabolism indicators. Transcriptome data from zebrafish showed a close correlation with steroid biosynthesis. The RNA-Sequencing was consistent with PCR results, indicating that CS downregulated gene (fdft1,lss,cyp51) expression concerned with steroid biosynthesis pathway in the diabetes model. CONCLUSION: CS effectively improved blood glucose levels, regulated glucose and lipid metabolism by suppressing gene expression in steroid biosynthesis pathway, and ameliorated hyperglycemia. Our research provides valuable insights for CS in the treatment of diabetes, and proposes a new strategy for selecting clinical medications for diabetes.

Porcine platelet lysates exert the efficacy of chondroregeneration and SMAD2-mediated anti-chondrofibrosis on knee osteoarthritis.

BACKGROUND: The lack of self-repairability in cartilage and the formation of fibrocartilage pose significant challenges in treating knee osteoarthritis, and there is still no ideal solution. Autologous platelet lysates have been clinically applied to treat kOA and exert satisfactory cartilage-repair efficacy, but the preparation of human PL brings damage to patients and is hardly standardized. METHODS: In this study, porcine PL was developed to replace hPL, and its chondroregenerative and anti-chondrofibrosis effects were explored. Enzyme-Linked Immunosorbent Assay was applied to qualify the PL products. In vivo, partial-thickness cartilage defects were created on rats as a kOA model, and the von Frey test, histopathological observation, immunohistochemical analysis, and western blot analysis were conducted. In vitro, CCK-8 assay, real-time PCR analysis, immunofluorescence test, and WB analysis were conducted for the mechanism study of pPL. RESULTS: The in vivo data showed that pPL significantly repaired the cartilage defect by improving matrix synthesis and also ameliorated the pain response in the kOA model of rats. In addition, pPL exerted an anti-fibrosis effect on cartilage by suppressing the expressions of COL1, COL3, α-SMA, VIMENTIN, SMAD2, p-SMAD2, and CTGF in cartilage. The in vitro data verified these effects and indicated that the SMAD2 pathway mediated the anti-fibrosis mechanism of pPL. Moreover, the comparable effects between pPL and rat PL indicate that there is no immune rejection from pPL. CONCLUSIONS: This study firstly demonstrated the anti-kOA effects of pPL on both cartilage-repair and anti-chondrofibrosis. It developed pPL as a promising alternative to autologous PL for clinical applications.

In Silico, In Vitro, and In Vivo Investigations on Adapalene as Repurposed Third Generation Retinoid against Multiple Myeloma and Leukemia.

The majority of hematopoietic cancers in adults are incurable and exhibit unpredictable remitting-relapsing patterns in response to various therapies. The proto-oncogene c-MYC has been associated with tumorigenesis, especially in hematological neoplasms. Therefore, targeting c-MYC is crucial to find effective, novel treatments for blood malignancies. To date, there are no clinically approved c-MYC inhibitors. In this study, we virtually screened 1578 Food and Drug Administration (FDA)-approved drugs from the ZINC15 database against c-MYC. The top 117 compounds from PyRx-based screening with the best binding affinities to c-MYC were subjected to molecular docking studies with AutoDock 4.2.6. Retinoids consist of synthetic and natural vitamin A derivatives. All-trans-retinoic acid (ATRA) were highly effective in hematological malignancies. In this study, adapalene, a third-generation retinoid usually used to treat acne vulgaris, was selected as a potent c-MYC inhibitor as it robustly bound to c-MYC with a lowest binding energy (LBE) of -7.27 kcal/mol, a predicted inhibition constant (pKi) of 4.69 µM, and a dissociation constant (Kd value) of 3.05 µM. Thus, we examined its impact on multiple myeloma (MM) cells in vitro and evaluated its efficiency in vivo using a xenograft tumor zebrafish model. We demonstrated that adapalene exerted substantial cytotoxicity against a panel of nine MM and two leukemic cell lines, with AMO1 cells being the most susceptible one (IC50 = 1.76 ± 0.39 µM) and, hence, the focus of this work. Adapalene (0.5 × IC50, 1 × IC50, 2 × IC50) decreased c-MYC expression and transcriptional activity in AMO1 cells in a dose-dependent manner. An examination of the cell cycle revealed that adapalene halted the cells in the G2/M phase and increased the portion of cells in the sub-G0/G1 phase after 48 and 72 h, indicating that cells failed to initiate mitosis, and consequently, cell death was triggered. Adapalene also increased the number of p-H3(Ser10) positive AMO1 cells, which is a further proof of its ability to prevent mitotic exit. Confocal imaging demonstrated that adapalene destroyed the tubulin network of U2OS cells stably transfected with a cDNA coding for α-tubulin-GFP, refraining the migration of malignant cells. Furthermore, adapalene induced DNA damage in AMO1 cells. It also induced apoptosis and autophagy, as demonstrated by flow cytometry and western blotting. Finally, adapalene impeded tumor growth in a xenograft tumor zebrafish model. In summary, the discovery of the vitamin A derivative adapalene as a c-MYC inhibitor reveals its potential as an avant-garde treatment for MM.

External Application of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Hyaluronic Acid Gel Repairs Foot Wounds of Types I and II Diabetic Rats Through Paracrine Action Mode.

Diabetic foot ulcer (DFU) is a main diabetic complication with unmet treatment needs. This study applied human umbilical cord-derived mesenchymal stem cells-hyaluronic acid (hucMSCs-HA) gel to treat DFU in a noninvasive external way and investigated its paracrine action and mechanism. In this study, after analyzing the physical and biological properties of HA gel, hucMSCs-HA gel was applied in 2 in vivo models (types I and II DFU), and a molecular mechanism was investigated. To evaluate the paracrine action of hucMSCs, hucMSCs-conditional medium (MSC-CM) was collected to treat 1 in vivo model (type I DFU) and 2 in vitro models (high glucose (HG)-injured human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts (HSFs)). The results indicated that HA gel with a porous microstructure underwent over 90% degradation and swelled to the maximum value within 48 h. In vivo, hucMSCs-HA gel accelerated wound healing of DFU rats by improving re-epithelialization, collagen deposition, and angiogenesis, in which a paracrine action of hucMSCs was confirmed and the phosphorylation of p38, ERK1/2, JNK, and Akt was increased. In vitro, MSC-CM improved cell viability, wound healing, migration, tube formation, cell senescence, and abnormal expressions (TNF-α, IL-1ß, IL-6, ET-1, p16 genes, and PCNA protein) of HUVECs, also improved cell viability, wound healing, antioxidant stress, and abnormal expressions (COL1, COL3, COL4, SOD1, SOD2 genes, and PCNA protein) of HSFs. Summarily, noninvasive external application of hucMSCs-HA gel shows great perspective against DFU and exerts wound healing effects through the MAPK and Akt pathways-mediated paracrine mechanism.

Integrating Network Pharmacology and Experimental Validation to Explore the Effects and Mechanisms of Qinghao Biejia Decoction and Its Active Compound Artemisinin B Against Non-Small-Cell Lung Cancer.

Purpose: To explore the pharmacological effects and mechanisms of Qinghao Biejia decoction (QBD) against non-small-cell lung cancer (NSCLC) based on network pharmacology and to verify the anticancer effect of artemisinin B (ART B), the active ingredient of QBD, on H1299 cells. Methods: Ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) was applied to explore the chemoprofile of QBD. A zebrafish xenograft model was used to determine the anti-cancer efficacy of QBD. Cell counting kit-8 assay, terminal deoxyribonucleotide transferase-mediated-dUTP nick-end labeling assay; immunofluorescence, and flow cytometry were used to evaluate the in vitro anti-proliferative and pro-apoptotic effects of QBD and ART B on H1299 cells. Subsequently, the related targets and action mechanisms of both QBD and ART B predicted by network pharmacological analyses were experimentally validated by real-time PCR and Western blot assays on H1299 cells. Results: UPLC-QTOF-MS/MS identified a total of 69 compounds (such as ART B, mangiferin, and artemisinic acid) in QBD. The in vivo data showed that QBD significantly inhibited the growth of H1299 cells in xenograft larval zebrafish from 125 to 500 µg/mL. The in vitro data showed that QBD induced apoptosis of H1299 cells, accompanied by down-regulating the expression of BCL-2 and up-regulating the expression of BIM, PUMA, BAX, c-PARP, γ-H2A.X, c-CASP3, and c-CASP8. Alike QBD, ART B exerted similar anti-proliferative and pro-apoptotic effects on H1299 cells. Moreover, ART B inhibited expressions of BCL2L1, AKT1, AKT2, MMP-2, and EGFR, and up-regulated ALB expression. Mechanistically, ART B promoted apoptosis of H1299 cells by inhibiting PI3K/Akt signaling pathway. Conclusion: This study revealed the anti-NSCLC efficacy of QBD. ART B, the effective component of QBD, plays an anti-NSCLC role by down-regulating the PI3K-Akt signaling pathway. It suggests that QBD and ART B are promising drug candidates for NSCLC treatment.

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects.

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

Gallic acid in theabrownin suppresses cell proliferation and migration in non­small cell lung carcinoma via autophagy inhibition.

The bioactive extract of green tea, theabrownin (TB), is known to exhibit pro-apoptotic and antitumor effects on non-small cell lung cancer (NSCLC). Gallic acid (GA) is a crucial component of TB; however, its mechanism of action in NSCLC has been rarely studied. To date, little attention has been paid to the anti-NSCLC activity of GA. Therefore, the present study investigated the effects of GA in vivo and in vitro. Cell Counting Kit (CCK)-8 assay, DAPI staining and flow cytometry, wound-healing assay and western blotting were used to assess cell viability, apoptosis, migration and protein expression, respectively. In addition, a xenograft model was generated, and TUNEL assay and immunohistochemistry analysis were performed. The CCK-8 data showed that the viability of H1299 cells was significantly inhibited by GA in a dose- and time-dependent manner. DAPI staining, Annexin-V/PI staining and wound-healing data showed that GA exerted pro-apoptotic and anti-migratory effects on H1299 cells in a dose-dependent manner. Furthermore, the results of western blotting showed that GA significantly upregulated the levels of pro-apoptotic proteins [cleaved (c-)PARP, c-caspase8, c-caspase-9 and the ratio of γ-H2A.X/H2A.X]. In vivo data confirmed the antitumor effect of GA through apoptosis induction in an autophagy-dependent manner. In conclusion, the present study confirmed the anti-proliferative, pro-apoptotic and anti-migratory effects of GA against NSCLC in vitro and in vivo, providing considerable evidence for its potential as a novel candidate for the treatment of NSCLC.

Detrimental alteration of mesenchymal stem cells by an articular inflammatory microenvironment results in deterioration of osteoarthritis.

BACKGROUND: Articular injection of mesenchymal stem cells (MSCs) has been applied to treat knee osteoarthritis (kOA), but its clinical outcomes are controversial. This study investigated whether an articular inflammatory microenvironment (AIM) impacts MSC-based therapy in a rat model of kOA. METHODS: The biological change of MSCs and the functional change of MSCs on chondrocytes were evaluated under AIM. The key mediator and mechanism for the AIM impact on MSC therapy were explored via gain- and loss-of-function approaches. RESULTS: The results showed that MSCs exerted potent anti-kOA effects in vivo and in vitro, but that this therapy become chondrodestructive if a chronic AIM was present. Mechanistically, the overexpression of MMP13 in the injected MSCs via a MAPKs-AP1 signaling axis was revealed as the underlying mechanism for the detriment outcome. CONCLUSIONS: This study thus clarifies recent clinical findings while also suggesting a means to overcome any detrimental effects of MSC-based therapy while improving its efficacy.

Cynaropicrin disrupts tubulin and c-Myc-related signaling and induces parthanatos-type cell death in multiple myeloma.

The majority of blood malignancies is incurable and has unforeseeable remitting-relapsing paths in response to different treatments. Cynaropicrin, a natural sesquiterpene lactone from the edible parts of the artichoke plant, has gained increased attention as a chemotherapeutic agent. In this study, we investigated the effects of cynaropicrin against multiple myeloma (MM) cells in vitro and assessed its in vivo effectiveness in a xenograft tumor zebrafish model. We showed that cynaropicrin exerted potent cytotoxicity against a panel of nine MM cell lines and two leukemia cell lines with AMO1 being the most sensitive cell line (IC50 = 1.8 ± 0.3 µM). Cynaropicrin (0.8, 1.9, 3.6 µM) dose-dependently reduced c-Myc expression and transcriptional activity in AMO1 cells that was associated with significant downregulation of STAT3, AKT, and ERK1/2. Cell cycle analysis showed that cynaropicrin treatment arrested AMO1 cells in the G2M phase along with an increase in the sub-G0G1 phase after 24 h. With prolonged treatment times, cells accumulated more in the sub-G0G1 phase, implying cell death. Using confocal microscopy, we revealed that cynaropicrin disrupted the microtubule network in U2OS cells stably expressing α-tubulin-GFP. Furthermore, we revealed that cynaropicrin promoted DNA damage in AMO1 cells leading to PAR polymer production by PARP1 hyperactivation, resulting in AIF translocation from the mitochondria to the nucleus and subsequently to a novel form of cell death, parthanatos. Finally, we demonstrated that cynaropicrin (5, 10 µM) significantly reduced tumor growth in a T-cell acute lymphoblastic leukemia (T-ALL) xenograft zebrafish model. Taken together, these results demonstrate that cynaropicrin causes potent inhibition of hematopoietic tumor cells in vitro and in vivo.

Intravenous injection of human umbilical cord-derived mesenchymal stem cells ameliorates not only blood glucose but also nephrotic complication of diabetic rats through autophagy-mediated anti-senescent mechanism.

BACKGROUND: Diabetic nephropathy (DN) is one of the most severe complications of diabetes mellitus, which is characterized by early occurrence of albuminuria and end-stage glomerulosclerosis. Senescence and autophagy of podocytes play an important role in DN development. Human umbilical cord-derived mesenchymal stem cells (hucMSCs) have potential in the treatment of diabetes and its complications. However, the role of hucMSCs in the treatment of DN and the underlying mechanism remain unclear. METHODS: In vivo, a streptozotocin-induced diabetic male Sprague Dawley rat model was established to determine the renoprotective effect of hucMSCs on DN by biochemical analysis, histopathology, and immunohistochemical staining of renal tissues. And the distribution of hucMSCs in various organs in rats within 168 h was analyzed. In vitro, CCK8 assay, wound healing assay, and ß-galactosidase staining were conducted to detect the beneficial effects of hucMSCs on high glucose-induced rat podocytes. Real-time PCR and western blot assays were applied to explore the mechanism of action of hucMSCs. RESULTS: The in vivo data revealed that hucMSCs were distributed into kidneys and significantly protected kidneys from diabetic damage. The in vitro data indicated that hucMSCs improved cell viability, wound healing, senescence of the high glucose-damaged rat podocytes through a paracrine action mode. Besides, the altered expressions of senescence-associated genes (p16, p53, and p21) and autophagy-associated genes (Beclin-1, p62, and LC3) were improved by hucMSCs. Mechanistically, hucMSCs protected high glucose-induced injury in rat podocytes by activating autophagy and attenuating senescence through the AMPK/mTOR pathway. CONCLUSIONS: In conclusion, hucMSCs might be a promising therapeutic strategy for the clinical treatment of DN-induced renal damages.

Nanofat lysate ameliorates pain and cartilage degradation of osteoarthritis through activation of TGF-ß-Smad2/3 signaling of chondrocytes.

Introduction: Nanofat is an effective cell therapy for osteoarthritis (OA). However, it has clinical limitations due to its short half-life. We developed Nanofat lysate (NFL) to overcome the defect of Nanofat and explore its anti-OA efficacy and mechanism. Methods: Monoiodoacetate (MIA) was employed to establish rat OA model. For pain assessment, paw withdrawal latency (PWL) and thermal withdrawal latency (TWL) were evaluated. Degeneration of cartilage was observed by histopathological and immunohistochemical examination. Primary chondrocytes were treated with TNF-α to establish the cellular model of OA. MTT, wound healing, and transwell assays were performed to assess effects of NFL on chondrocytes. RNA-seq, qPCR and Western blot assays were conducted to clarify the mechanism of NFL. Results and Discussion: The animal data showed that PWL and TWL values, Mankin's and OARSI scorings, and the Col2 expression in cartilage were significantly improved in the NFL-treated OA rats. The cellular data showed that NFL significantly improved the proliferation, wound healing, and migration of chondrocytes. The molecular data showed that NFL significantly restored the TNF-α-altered anabolic markers (Sox9, Col2 and ACAN) and catabolic markers (IL6 and Mmp13). The RNA-seq identified that TGF-ß-Smad2/3 signaling pathway mediated the efficacy of NFL, which was verified by qPCR and Western blot that NFL significantly restored the abnormal expressions of TGFßR2, phosphorylated-Smad2, phosphorylated-Smad2/3, Col2, Mmp13 and Mmp3. After long-term storage, NFL exerted similar effects as its fresh type, indicating its advantage of storability. In sum, NFL was developed as a new therapeutic approach and its anti-OA efficacy and mechanism that mediated by TGF-ß-Smad2/3 signaling was determined for the first time. Besides, the storability of NFL provided a substantial advantage than other living cell-based therapies.

An Innovative Model of ISS-Based Multiple Fractures and Gastrointestinal Dysfunction Related to c-Kit Protein Expression on Interstitial Cells of Cajal.

OBJECTIVE: Gastrointestinal dysfunction seriously affects the prognosis and quality of life of patients with multiple fractures. However, experimental evidence of this relationship is lacking. Here we describe a newly developed mouse model of postoperative gastrointestinal dysfunction after multiple fractures. METHODS: Trauma severity was assessed using the injury severity score (ISS). Based on the ISS, a multiple fracture model was established in mice as follows: limb fractures with pelvic fractures and multiple rib fractures; limb fractures with multiple rib fractures; closed fracture of both forelegs with pelvic fracture and rib fractures; closed limb fractures; limb fracture with pelvic fracture; spinal fractures; hind leg fractures with pelvic fractures; pelvic fracture with multiple rib fractures; closed fracture of both fore legs with pelvic fracture; and closed fracture of both fore legs with multiple rib fractures. In each model group, gastrointestinal motility was assayed and the histopathology of the small intestine was examined. Western blot and immunohistochemical analyses of jejunal tissue were performed to detect c-kit protein expression, the level of which was compared with that of a control group. The results of ANOVA are expressed as mean ± standard deviation. RESULTS: In mice with multiple fractures, food intake was greatly reduced, consistent with histopathological evidence of an injured intestinal epithelium. The jejunal tissue of mice in groups a, c, f, and h was characterized by extensively necrotic and exfoliated intestinal mucosal epithelium and inflammatory cell infiltration in the lamina propria. In the gastrointestinal function assay, gastrointestinal motility was significantly reduced in groups a, b, c, f, and g; these group also had a higher ISS (p < 0.01). The expression of c-kit protein in groups with gastrointestinal dysfunction was significantly up-regulated (p < 0.001) compared with the control group. The close correlation between c-kit expression and the ISS indicated an influence of trauma severity on gastrointestinal motility. CONCLUSION: Gastrointestinal dysfunction after multiple fractures was successfully reproduced in a mouse model. In these mice, c-kit expression correlated with gastrointestinal tissue dysfunction and might serve as a therapeutic target.

Acute Developmental Toxicity of Panax notoginseng in Zebrafish Larvae.

OBJECTIVE: To evaluate toxicity of raw extract of Panax notoginseng (rPN) and decocted extract of PN (dPN) by a toxicological assay using zebrafish larvae, and explore the mechanism by RNA sequencing assay. METHODS: Zebrafish larvae was used to evaluate acute toxicity of PN in two forms: rPN and dPN. Three doses (0.5, 1.5, and 5.0 µ g/mL) of dPN were used to treat zebrafishes for evaluating the developmental toxicity. Behavior abnormalities, body weight, body length and number of vertebral roots were used as specific phenotypic endpoints. RNA sequencing (RNA-seq) assay was applied to clarify the mechanism of acute toxicity, followed by real time PCR (qPCR) for verification. High performance liquid chromatography analysis was performed to determine the chemoprofile of this herb. RESULTS: The acute toxicity result showed that rPN exerted higher acute toxicity than dPN in inducing death of larval zebrafishes (P<0.01). After daily oral intake for 21 days, dPN at doses of 0.5, 1.5 and 5.0 µ g/mL decreased the body weight, body length, and vertebral number of larval zebrafishes, indicating developmental toxicity of dPN. No other adverse outcome was observed during the experimental period. RNA-seq data revealed 38 genes differentially expressed in dPN-treated zebrafishes, of which carboxypeptidase A1 (cpa1) and opioid growth factor receptor-like 2 (ogfrl2) were identified as functional genes in regulating body development of zebrafishes. qPCR data showed that dPN significantly down-regulated the mRNA expressions of cpa1 and ogfrl2 (both P<0.01), verifying cpa1 and ogfrl2 as target genes for dPN. CONCLUSION: This report uncovers the developmental toxicity of dPN, suggesting potential risk of its clinical application in children.

Intra-articular injection of placental mesenchymal stromal cells ameliorates pain and cartilage anabolism/catabolism in knee osteoarthritis.

Background: Knee Osteoarthritis (kOA), the most common joint degenerative disorder, lacks effective therapeutics. Placenta-derived mesenchymal stromal cells (PMSCs) are effective in tissue repairing and generation, which have potential in treating kOA. This study aimed to determine the anti-kOA efficacy of PMSCs and to explore its action mode. Methods: Flow cytometry and three-line differentiation were performed for identification of PMSCs. In vivo, a rat kOA model established by anterior cruciate ligament transection (ACLT) surgery was used to evaluate the efficacy of PMSCs. Histopathological HE and SO staining with Osteoarthritis Research Society International scoring were conducted, and cartilage expressions of MMP13 and Col2 were measured by immunohistochemistry. Pain behavior parameters by mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL), were measured. In vitro, wound healing and cell immunofluorescence assays were conducted to detect the proliferation and migration ability of chondrocytes treated with PMSCs conditioned medium (PMSCs-CM). Quantitative real-time PCR (qRT-PCR) and Western blot (WB) assays were applied to explore the molecular action of PMSCs on chondrocytes. Results: The results of flow cytometry indicated that the surface markers of PMSCs (CD73 > 95%, CD90 > 95%, and CD34 < 2%) were consistent with the typical mesenchymal stromal cells. The in vivo data showed that PMSCs significantly reversed the kOA progression by protection of cartilage, regulation of anabolic (Col2) and catabolic (MMP13) expressions, and relief of pain symptoms. The in vitro data showed that PMSCs promoted chondrocyte proliferation and migration and significantly restored the IL-1ß-induced abnormal gene expressions of Col2, Mmp13, Adamts4, Adamts5 and Sox9 and also restored the abnormal protein expressions of Col2, Mmp13 and Sox9 of chondrocytes. The molecular actions of PMSCs on chondrocytes in nested co-culture way or in conditioned medium way were similar, confirming a paracrine-based mode of action. Conclusion: This study demonstrated PMSCs' anti-kOA efficacy and its paracrine-based action mode, providing novel knowledge of PMSCs and suggesting it as a promising cell therapy for treatment of kOA.

Identification of Gedunin from a Phytochemical Depository as a Novel Multidrug Resistance-Bypassing Tubulin Inhibitor of Cancer Cells.

The chemotherapy of tumors is frequently limited by the development of resistance and severe side effects. Phytochemicals may offer promising candidates to meet the urgent requirement for new anticancer drugs. We screened 69 phytochemicals, and focused on gedunin to analyze its molecular modes of action. Pearson test-base correlation analyses of the log10IC50 values of 55 tumor cell lines of the National Cancer Institute (NCI), USA, for gedunin with those of 91 standard anticancer agents revealed statistically significant relationships to all 10 tested microtubule inhibitors. Thus, we hypothesized that gedunin may be a novel microtubule inhibitor. Confocal microscopy, cell cycle measurements, and molecular docking in silico substantiated our assumption. Agglomerative cluster analyses and the heat map generation of proteomic data revealed a subset of 40 out of 3171 proteins, the expression of which significantly correlated with sensitivity or resistance for the NCI cell line panel to gedunin. This indicates the complexity of gedunin's activity against cancer cells, underscoring the value of network pharmacological techniques for the investigation of the molecular modes of drug action. Finally, we correlated the transcriptome-wide mRNA expression of known drug resistance mechanism (ABC transporter, oncogenes, tumor suppressors) log10IC50 values for gedunin. We did not find significant correlations, indicating that gedunin's anticancer activity might not be hampered by classical drug resistance mechanisms. In conclusion, gedunin is a novel microtubule-inhibiting drug candidate which is not involved in multidrug resistance mechanisms such as other clinically established mitotic spindle poisons.

Onset of p53/NF-κB signaling crosstalk in human melanoma cells in response to anti-cancer theabrownin.

As a major tea component, theabrownin represents a promising anti-cancer candidate. However, its effect on the melanoma is unknown. To evaluate the in vitro and in vivo anti-melanoma efficacy of TB, we conducted cell viability, immunostaining, comet, and TUNEL assays on human A375 melanoma cells, and employed a zebrafish xenograft model of A375 cells. Real-time PCR (qPCR) and western blot were conducted to explore the molecular mechanisms of TB. In vitro, TB significantly inhibited the proliferation of A375 cells, and A375 cells showed the highest inhibitory rate among the other melanoma cell line (A875) and human dermal fibroblasts. TB triggered DNA damage and induced apoptosis of A375 cells and significantly inhibited the growth of A375 xenograft tumors in zebrafishes. Several key molecular events were activated by TB, including DNA damage-associated p53 and NF-κB pathways, through up-regulation of GADD45α, γ-H2A.X, phospho-ATM(p-ATM), phospho-ATR (p-ATR), phospho-p53 (p-p53), phospho-IKKα/ß (p-IKKα/ß), phospho-p65 (p-p65), etc. However, the TB-activated molecular events were counteracted by either knockdown of p53 or p65, and only dual knockdown of both p53 and p65 completed counteracted the anti-melanoma efficacy of TB. In conclusion, TB triggered DNA damage and thereby inhibited proliferation and induced cellular senescence and apoptosis of melanoma cells through mechanisms mediated by p53/NF-κB signaling crosstalk. This is the first report on the efficacy and mechanisms of TB on melanoma cells, making TB a promising candidate for anti-melanoma agent development.