Discovery of novel peptide-dehydroepiandrosterone hybrids inducing endoplasmic reticulum stress with effective in vitro and in vivo anti-melanoma activities.

Steroid hybrids have emerged as a type of advantageous compound as they could offer improved pharmacological and pharmaceutical properties. Here, we report a series of novel peptide-dehydroepiandrosterone hybrids, which would effectively induce endoplasmic reticulum stress (ERS) and lead to apoptosis with outstanding in vitro and in vivo anti-melanoma effects. The lead compound IId among various steroids conjugated with peptides and pyridines showed effective in vivo activity in B16 xenograft mice: in medium- and high-dose treatment groups (60 and 80 mg/kg), compound IId would significantly inhibit the growth of tumours by 98%-99% compared to the control group, with the highest survival rate as well. Further mechanism studies showed that compound IId would damage the endoplasmic reticulum and upregulate the ERS markers C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78), which could further regulate caspase and Bcl-2 family proteins and lead to cell apoptosis. The compound IId was also proven to be effective in inhibiting B16 cell migration and invasion.

Andrographolide sensitizes KRAS-mutant colorectal cancer cells to cetuximab by inhibiting the EGFR/AKT and PDGFRß/AKT signaling pathways.

BACKGROUND: Cetuximab, an inhibitor targeting EGFR, is widely applied in clinical management of colorectal cancer (CRC). Nevertheless, drug resistance induced by KRAS-mutations limits cetuximab's anti-cancer effectiveness. Furthermore, the persistent activation of EGFR-independent AKT is another significant factor in cetuximab resistance. Nevertheless, the mechanism that EGFR-independent AKT drives cetuximab resistance remains unclear. Thus, highlighting the need to optimize therapies to overcome cetuximab resistance and also to explore the underlying mechanism. PURPOSE: This work aimed to investigate whether and how andrographolide enhance the therapeutic efficacy of cetuximab in KRAS-mutant CRC cells by modulating AKT. METHODS: The viabilities of CRC cell lines were analyzed by CCK-8. The intracellular proteins phosphorylation levels were investigated by Human Phospho-kinase Antibody Array analysis. Knockdown and transfection of PDGFRß were used to evaluate the role of andrographolide on PDGFRß. The western blotting was used to investigate Wnt/ß-catenin pathways, PI3K/AKT, and EMT in KRAS-mutant CRC cells. The animal models including subcutaneous tumor and lung metastasis were performed to assess tumor response to therapy in vivo. RESULTS: Andrographolide was demonstrated to decrease the expression of PI3K and AKT through targeting PDGFRß and EGFR, and it enhanced cetuximab effect on KRAS-mutant CRC cells by this mechanism. Meanwhile, andrographolide helped cetuximab to inhibit Wnt/ß-catenin, CRC cell migration and reduced Vimentin expression, while increasing that of E-cadherin. Lastly, co-treatment with cetuximab and andrographolide reduced the growth of KRAS-mutant tumors and pulmonary metastases in vivo. CONCLUSIONS: Our findings suggest that andrographolide can overcome the KRAS-mutant CRC cells' resistance to cetuximab through inhibiting the EGFR/PI3K/AKT and PDGFRß /AKT signaling pathways. This research provided a possible theory that andrographolide sensitizes KRAS-mutant tumor to EGFR TKI.

Novel 3D-printing bilayer GelMA-based hydrogel containing BP,ß-TCP and exosomes for cartilage-bone integrated repair.

The integrated repair of cartilage and bone involves the migration and differentiation of cells, which has always been a difficult problem to be solved. We utilize the natural biomaterial gelatin to construct gelatin methacryloyl (GelMA), a hydrogel scaffold with high cell affinity. GelMA is mixed with different components to print a bi-layer porous hydrogel scaffold with different modulus and composition in upper and lower layers through three-dimensional (3D) printing technology. The upper scaffold adds black phosphorus (BP) and human umbilical cord mesenchymal stem cells (hUMSCs) exosomes (exos) in GelMA, which has a relatively lower elastic modulus and is conducive to the differentiation of BMSCs into cartilage. In the lower scaffold, in addition to BP and hUMSCs exos,ß-tricalcium phosphate (ß-TCP), which has osteoconductive and osteoinductive effects, is added to GelMA. The addition ofß-TCP significantly enhances the elastic modulus of the hydrogel scaffold, which is conducive to the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).In vitroexperiments have confirmed that the bi-layer scaffolds can promote osteogenesis and chondrogenic differentiation respectively. And in the rabbit cartilage-bone injury model, MRI and micro-CT results show that the 3D printed bi-layer GelMA composite scaffold has a repair effect close to normal tissue.

Marsdenia tenacissima enhances immune response of tumor infiltrating T lymphocytes to colorectal cancer.

Introduction: Tumor-infiltrating T lymphocytes in the tumor microenvironment are critical factors influencing the prognosis and chemotherapy outcomes. As a Chinese herbal medicine, Marsdenia tenacissima extract (MTE) has been widely used to treat cancer in China. Its immunoregulatory effects on tumor-associated macrophages is well known, but whether it regulates tumor-infiltrating T-cell functions remains unclear. Method: We collected 17 tumor samples from MTE-administered colorectal cancer patients, 13 of which showed upregulation of CD3+/CD8+ tumor-infiltrating T cells. Further in vitro and in vivo experiments were performed to investigate the regulatory effects of MTE on tumor-infiltrating T cells and immune escape of tumors. Results: Under single and co-culture conditions, MTE inhibited TGF-ß1 and PD-L1 expression in the colorectal cancer (CRC) cell lines HCT116 and LoVo. In Jurkat cells, MTE inhibited FOXP3 and IL-10 expression, increased IL-2 expression, but had no effect on PD-1 expression. These findings were confirmed in vitro using subcutaneous and colitis-associated CRC mouse models. MTE also increased the density of CD3+/CD8+ tumor-infiltrating T cells and exhibited considerable tumor-suppressive effects in these two tumor mouse models. Conclusions: Our findings suggested that MTE inhibits the immune escape of cancer cells, a precipitating factor increasing the immune response of T lymphocytes.

Platycodin D represses ß-catenin to suppress metastasis of cetuximab-treated KRAS wild-type colorectal cancer cells.

Cetuximab, an epidermal growth factor receptor (EGFR) inhibitor, is extensively used for clinical therapy in KRAS wild-type colorectal cancer (CRC) patients. However, some patients still cannot get benefit from the therapy, because metastasis and resistance occur frequently after cetuximab treatment. New adjunctive therapy is urgently needed to suppress metastasis of cetuximab-treated CRC cells. In this study, we used two KRAS wild-type CRC cells, HT29 and CaCo2, to investigate whether platycodin D, a triterpenoid saponin isolated from Chinese medicinal herb Platycodon grandifloras, is able to suppress the metastasis of cetuximab-treated CRC. Label-free quantitative proteomics analyses showed that platycodin D but not cetuximab significantly inhibited expression of ß-catenin in both CRC cells, and suggested that platycodin D counteracted the inhibition effect of cetuximab on cell adherence and functioned in repressing cell migration and invasion. Western blot results showed that single platycodin D treatment or combined platycodin D and cetuximab enhanced inhibition effects on expressions of key genes in Wnt/ß-catenin signaling pathway, including ß-catenin, c-Myc, Cyclin D1 and MMP-7, compared to single cetuximab treatment. Scratch wound-healing and transwell assays showed that platycodin D combined with cetuximab suppressed migration and invasion of CRC cells, respectively. Pulmonary metastasis model of HT29 and CaCo2 in nu/nu nude mice consistently showed that combined treatment using platycodin D and cetuximab inhibited metastasis significantly in vivo. Our findings provide a potential strategy to inhibit CRC metastasis during cetuximab therapy by addition of platycodin D.

Effect of a high vs. standard dose of vitamin D3 supplementation on bone metabolism and kidney function in children with chronic kidney disease.

We investigated the effects of high- vs. standard-dose vitamin D supplementation on kidney function and bone metabolism in children with chronic kidney disease (CKD). Children were randomized to receive one of two formulations: 75 participants received 2,000 IU/D of oral supplementation of vitamin D, while 75 participants received 400 IU/d for a minimum of 4 months. We investigated the effects of vitamin D supplementation on kidney-related indicators and bone metabolism-related indicators at different doses. A total of 158 participants were screened, among whom 150 met the inclusion criteria. The indicators of chronic kidney disease such as eGFR and serum uric acid were negatively correlated with the 25(OH)D level and BMD. Serum 25(OH)D and osteocalcin levels were positively correlated with spine BMD. The standard dose of vitamin D can improve the serum uric acid level, but high doses of vitamin D supplementation had no significant effect on the serum uric acid level. High doses of vitamin D supplementation can also improve the alkaline phosphatase level. When comparing the results of different doses of vitamin D supplementation, it was found that high-dose vitamin D supplementation did not improve bone density in the spine and femur neck relative to the standard dose of vitamin D but improved hypocalcemia and N-terminal propeptide of the human procollagen type I (PINP) level. Among the children with clinical kidney disease, high-dose vitamin D treatment for 4 months resulted in statistically significant improvement in kidney function but no significant difference in bone metabolism compared with the standard-dose vitamin D treatment.

Ginseng-Astragalus-oxymatrine injection ameliorates cyclophosphamide-induced immunosuppression in mice and enhances the immune activity of RAW264.7 cells.

ETHNOPHARMACOLOGICAL SIGNIFICANCE: Ginseng quinquefolium (L.), Astragalus membranaceus, and Sophora flavescens Aiton are popular folk medicines in many Asian countries and regions. These three traditional Chinese herbs and their extracts have been reported to considerably enhance the immune function. G. quinquefolium (L.) is considered the king of herbs in China. Traditionally, G. quinquefolium (L.) is believed to replenish vitality, which is considered as immune enhancement in modern Chinese pharmacy. One of the main uses of Astragalus is immunity enhancement; S. flavescens and oxymatrine obtained from its extract have been used to treat leukopenia. Considering the pharmacological properties of Ginseng, Astragalus, and oxymatrine, we evaluated the immunopotentiation effects of their combination, Ginseng-Astragalus-oxymatrine (GAO), in the present study. AIM OF THE STUDY: This study aimed to expand the clinical application of GAO and to preliminarily explore its mechanism of action by determining whether GAO injection can enhance immunity in vivo and in vitro. METHODS: Overall, 17 major chemical components in GAO were analysed using HPLC and LC-MS. The immunity-enhancing effect of GAO was studied in the cyclophosphamide (CTX)-induced immunosuppressive mouse model and RAW 264.7 cells. RESULTS: Quantitative analysis showed that the potential active components of GAO include at least ginsenosides, astragaloside IV, and oxymatrine. GAO could significantly improve the nonspecific immunity including the indices of the thymus and spleen, number of peripheral blood leukocytes, levels of TNF-α and IL-6, phagocytic function of macrophages, and cytotoxic activity of natural killer (NK) cells. Additionally, GAO enhanced the humoural immunity, characterised by the antibody production ability of B cells, and cellular immunity, characterised by the activity of T cells, in immunosuppressed mouse. Moreover, GAO could enhance the phagocytic and adhesion functions of RAW 264.7 cells, which may be related to the activation of reactive oxygen species and NF-κB signalling pathway. CONCLUSION: GAO could dramatically ameliorate CTX-induced immunosuppression in mouse and stimulate the immune activity in RAW 264.7 cells possibly by activating the NF-κB signalling pathway.

Effect of Shenfu Injection on Isolated Empty Beating Hearts from Miniature Pigs.

OBJECTIVE: To investigate the effect of Shenfu (SF) injection on donor heart preservation. METHODS: Twelve pigs were randomly divided into SF group (n=6) and control group (n=6). After eight hours of perfusion, the differences in hemoglobin, the expression of Bcl-2 and BAX, and changes in the myocardial ultrastructure were compared to illustrate the effects of SF injection in heart preservation. RESULTS: The differences in free hemoglobin between the SF group and the control group were statistically significant (P=0.001), and there was significant interaction of groups with times (P=0.019), but the perfusion time may not be associated with the hemoglobin concentration (P=0.616). According to Western blotting analysis, the expression of Bcl-2 was higher in the SF group than in the control group, while the expression of BAX was not different between the two groups. As to ultrastructural changes, both groups exhibited mitochondrial swelling and myofilament lysis, but the degree of damage in the SF group was smaller. CONCLUSION: Our study suggests that the application of SF injection for heart preservation may protect against cardiomyocytes and erythrocytes apoptosis, and Bcl-2 protein may play a role in these physiological processes.

Effect of shenfu injection on isolated empty beating hearts from miniature pigs

Abstract Objective: To investigate the effect of Shenfu (SF) injection on donor heart preservation. Methods: Twelve pigs were randomly divided into SF group (n=6) and control group (n=6). After eight hours of perfusion, the differences in hemoglobin, the expression of Bcl-2 and BAX, and changes in the myocardial ultrastructure were compared to illustrate the effects of SF injection in heart preservation. Results: The differences in free hemoglobin between the SF group and the control group were statistically significant (P=0.001), and there was significant interaction of groups with times (P=0.019), but the perfusion time may not be associated with the hemoglobin concentration (P=0.616). According to Western blotting analysis, the expression of Bcl-2 was higher in the SF group than in the control group, while the expression of BAX was not different between the two groups. As to ultrastructural changes, both groups exhibited mitochondrial swelling and myofilament lysis, but the degree of damage in the SF group was smaller. Conclusion: Our study suggests that the application of SF injection for heart preservation may protect against cardiomyocytes and erythrocytes apoptosis, and Bcl-2 protein may play a role in these physiological processes.

Strontium Alleviates Endoplasmic Reticulum Stress in a Nonalcoholic Fatty Liver Disease Model.

The purpose of this study was to explore the effects of strontium on fatty liver, and to clarify the possible mechanisms by which strontium improves nonalcoholic fatty liver disease (NAFLD). We also evaluated how strontium affected the endoplasmic reticulum stress (ERS) pathways. We established an in vitro model of NAFLD using a human hepatocyte cell line (L02) treated with 0.2 mM palmitic acid. The Sprague-Dawley rats were fed with a high-fat diet (HFD) to establish NAFLD model in vivo. After strontium treatment, the total cholesterol (TC), triglyceride (TG), and lipid deposition in L02 cells and liver tissues were determined. Strontium treatment suppressed intracellular TC and TG levels and lipid accumulation in L02 cells, and the effect of high concentrations of strontium were more obvious. Strontium significantly reduced the mRNA and protein expression of glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), inositol requiring enzyme 1 (IRE1), SREBP cleavage activator protein (SCAP), sterol regulatory element binding protein 1c (SREBP-1c), and SREBP-2 in L02 cells. In HFD-fed rats, strontium treatment reduced serum TC, TG, and low density lipoprotein cholesterol (LDL-C) levels, concurrent with a decrease in hepatic lipid accumulation. Furthermore, strontium treatment reduced the expression of GRP78 and SREBP-2 protein in liver tissues. Overall, strontium alleviated hepatic steatosis by decreasing ERS-related protein expression in vivo and in vitro models. The results indicated that strontium has the potential to become a new therapy for the prevention and treatment of NAFLD.

Comparative research on stability of baicalin and baicalein administrated in monomer and total flavonoid fraction form of Radix scutellariae in biological fluids in vitro.

CONTEXT: Baicalin (BL) and baicalein (B) as the major flavonoids of Scutellaria baicalensis Georgi (Lamiaceae) have been investigated intensively, and shown to possess a multitude of pharmacological activities. OBJECTIVE: This study systematically evaluates the stability of BL and B in monomer and total flavonoid fraction (FSR) form in vitro, and further studies whether the protective measures are effective to make B and BL stable enough to meet the requirement of quantitative analysis in various biological samples. MATERIALS AND METHODS: The stability of BL and B was evaluated by investigating the influence factors such as pH (2.0, 3.0, 4.5, 6.8, 7.4 and 9.0), temperature (4, 25 and 40 °C), antioxidant (vitamin C and Na2SO3) and sunlight. After the protective measures were taken, stability of BL and B in plasma, urine and tissue homogenates was evaluated through post-preparative stability (stored at 4 °C for 24 h), three freeze-thaw cycles stability and long-term stability test (stored in refrigerator at -20 °C for 15 days). In addition, by comparing the degradation parameters of BL and B obtained from the monomer administration group with those of the FSR administration group, drug-drug interaction of coexistent components in FSR on the stability of BL and B was discussed. RESULTS: The degradation of BL and B was both pH- and temperature-dependent with their correlation coefficents for first-order kinetics equation larger than 0.99, and acidic environment (pH 2-4.5), lower temperature (<4 °C) and acidic antioxidant (e.g. vitamin C) were conducive to stabilize B and BL. Furthermore, coexistent components in FSR were proved to have function on inhibiting the degradation of BL and B in our study for the first time, which was characteristic of prolonging their biological half-life (t1/2) significantly, e.g., from 2.89 h to indefinite for BL (pH 6.8, 25 °C), from 2.63 h to 4.48 h for B (pH 6.8, 25 °C) and so on. Antioxidant of Na2SO3 could inhibit the degradation of BL with t1/2 increasing from 1.8 h to 3.5 h, but aggravate the bio-transformation of B with t1/2 decreasing from 0.92 h to 0.29 h. Our research proved that BL monomer, and BL and B in FSR form could be stabilized enough to meet the requirement of biological quantitative analysis under the protection of coexistent components in FSR. DISCUSSION AND CONCLUSION: The results obtained indicated that the stability of BL and B was affected not only by its environmental parameters, but also by the coexistent components in the effective total flavonoids fractions.

Tanshinone IIA attenuates bleomycin-induced pulmonary fibrosis in rats.

Idiopathic pulmonary fibrosis is a chronic and progressive fibrotic lung disorder with unknown etiology and a high mortality rate. Tanshinone â…¡A (Tan â…¡A) is a lipophilic diterpene extracted from the Chinese herb Salvia miltiorrhiza Bunge with diverse biological functions. The present study was conducted to evaluate the effects of Tan â…¡A on bleomycin (BLM)­induced pulmonary fibrosis in rats. Rats received an intraperitoneal injection of Tan â…¡A and normal rats were used as controls. Severe pulmonary edema, inflammation and fibrosis were observed in the BLM­treated rats and the counts of total cells, neutrophils and lymphocytes were significantly increased in the bronchoalveolar lavage fluids of those rats. These pathological changes were markedly attenuated by subsequent treatment with Tan â…¡A. In addition, BLM­induced increased expression of tumor necrosis factor­α, interleukin (IL)­1ß, IL­6, cyclooxygenase­2, prostaglandin E2, malondialdehyde, inducible nitric oxide synthase and nitric oxide in rats, which was also suppressed by Tan â…¡A injection. The present findings suggest therapeutic potential of Tan â…¡A for pulmonary fibrosis.

[Absorption mechanism of oxysophocarpine across Caco-2 cell monolayer mode].

OBJECTIVE: To investigate the absorption mechanism of oxysophocarpine across Caco-2 cell monolayer model. METHOD: The safety concentration of oxysophocarpine in Caco-2 cell was first selected by using MTT method. Then the Caco-2 cell monolayers drug transport model was assigned to study the bi-direction transport mechanism of oxysophocarpine by evaluating the influent factors such as time, concentration, pH, P-gp inhibitor of verapamil, on its absorption characterization. RESULT: In the Caco-2 cell monolayer model, the transport volume was correlated positively with the time and concentration of oxysophocarpine, and affected by pH value. Verapamil had no influence on its transport since the transport of oxysophocarpine from apical (AP) to basolateral (BL) was similar to the transport from basolateral to apical. CONCLUSION: The intestinal absorption mechanism of oxysophocarpine was deduced as passive transference by Caco-2 cell monolayer model.

Synthesis and activity in enhancing long-term potentiation (LTP) of clausenamide stereoisomers.

Clausenamide, isolated from aqueous extract of dry leaves of Clausena lansium, a Chinese folk medicine, was found to have potent activity in enhancing LTP and show nootropic activity in animal tests. In order to discovery more potent stereoisomers and to analyze the relationship of structure-activity, the synthesis of 16 (8 pairs) optically pure stereoisomers of clausenamide with four chiral centers was achieved. The results of LTP assay showed that the nootropic activity of the stereoisomers of clausenamide is closely related to the configuration of stereoisomers.