[Effect of Chlorambucil Combined with Ibrutinib on Mantle Cell Lymphoma Cell Line Jeko-1 and Its Related Mechanism].

OBJECTIVE: To investigate the toxic effect of chlorambucil combined with ibrutinib on mantle cell lymphoma (MCL) cell line Jeko-1 and its related mechanism. METHODS: The MCL cell line Jeko-1 was incubated with different concentrations of chlorambucil or ibrutinib or the combination of the two drugs, respectively. CCK-8 assay was used to detect the proliferation of the cells, and Western blot was used to measure the protein expression levels of BCL-2, caspase-3, PI3K, AKT and P-AKT. RESULTS: After Jeko-1 cells were treated with chlorambucil (3.125, 6.25, 12.5, 25, 50 µmol/L) and ibrutinib (3.125, 6.25, 12.5, 25, 50 µmol /L) alone for 24, 48, 72h respectively, the cell proliferation was inhibited in a time- and dose-dependent manner. Moreover, the two drugs were applied in combination at low doses (single drug inhibition rate<50%), and the results showed that the combination of two drugs had a more significant inhibitory effect (all P < 0.05). Compared with the control group, the apoptosis rate of the single drug group of chlorambucil (3.125, 6.25, 12.5, 25, 50 µmol/L) and ibutinib (3.125, 6.25, 12.5, 25, 50 µmol/L) was increased in a dose-dependent manner. The combination of the two drugs at low concentrations (3.125, 6.25, 12.5 µmol/L) could significantly increase the apoptosis rate compared with the corresponding concentration of single drug groups (all P < 0.05). Compared with control group, the protein expression levels of caspase-3 in Jeko-1 cells were upregulated, while the protein expression levels of BCL-2, PI3K, and p-AKT/AKT were downregulated after treatment with chlorambucil or ibrutinib alone. The combination of the two drugs could produce a synergistic effect on the expressions of the above-mentioned proteins, and the differences between the combination group and the single drug groups were statistically significant (all P < 0.05). CONCLUSION: Chlorambucil and ibrutinib can promote the apoptosis of MCL cell line Jeko-1, and combined application of the two drugs shows a synergistic effect, the mechanism may be associated with the AKT-related signaling pathways.

Bidirectional Regulation of Sodium Acetate on Macrophage Activity and Its Role in Lipid Metabolism of Hepatocytes.

Short-chain fatty acids (SCFAs) are important metabolites of the intestinal flora that are closely related to the development of non-alcoholic fatty liver disease (NAFLD). Moreover, studies have shown that macrophages have an important role in the progression of NAFLD and that a dose effect of sodium acetate (NaA) on the regulation of macrophage activity alleviates NAFLD; however, the exact mechanism of action remains unclear. This study aimed to assess the effect and mechanism of NaA on regulating the activity of macrophages. RAW264.7 and Kupffer cells cell lines were treated with LPS and different concentrations of NaA (0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, and 5 mM). Low doses of NaA (0.1 mM, NaA-L) significantly increased the expression of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin 1 beta (IL-1ß); it also increased the phosphorylation of inflammatory proteins nuclear factor-κB p65 (NF-κB p65) and c-Jun (p < 0.05), and the M1 polarization ratio of RAW264.7 or Kupffer cells. Contrary, a high concentration of NaA (2 mM, NaA-H) reduced the inflammatory responses of macrophages. Mechanistically, high doses of NaA increased intracellular acetate concentration in macrophages, while a low dose had the opposite effect, consisting of the trend of changes in regulated macrophage activity. Besides, GPR43 and/or HDACs were not involved in the regulation of macrophage activity by NaA. NaA significantly increased total intracellular cholesterol (TC), triglycerides (TG), and lipid synthesis gene expression levels in macrophages and hepatocytes at either high or low concentrations. Furthermore, NaA regulated the intracellular AMP/ATP ratio and AMPK activity, achieving a bidirectional regulation of macrophage activity, in which the PPARγ/UCP2/AMPK/iNOS/IκBα/NF-κB signaling pathway has an important role. In addition, NaA can regulate lipid accumulation in hepatocytes by NaA-driven macrophage factors through the above-mentioned mechanism. The results revealed that the mode of NaA bi-directionally regulating the macrophages further affects hepatocyte lipid accumulation.

Wavefront aberrations caused by biomechanical effects after Small Incision Lenticule Extraction (SMILE) based on finite element analysis.

To examine wavefront aberrations induced by biomechanical effects after Small Incision Lenticule Extraction (SMILE) surgery. The three-dimensional (3D) finite element models of the human eye were established. By loading the intraocular pressure (IOP), the displacement of the anterior and posterior surface of the cornea was calculated. Then the displacement was converted into the wavefront aberrations by wave-surface fitting. The results showed that the induced wavefront aberrations were noticeable from biomechanical effects after SMILE surgery. The induced higher-order aberrations from the anterior corneal surface included spherical aberration, y-Trefoil, and x-Tetrafoil. Spherical aberration was positively correlated with corrected diopter (D), but x-Tetrafoil and y-Trefoil remained stable. The induced wavefront aberrations from the posterior corneal surface were smaller than those from the anterior corneal surface, and some of the aberrations compensated each other. With IOP increased, defocus and x-Tetrafoil from the anterior corneal surface increased, while y-Trefoil and spherical aberration decreased. The IOP only affected defocus from the posterior corneal surface. In addition, the incision size also had a distinct impact on primary x-astigmatism and x-Trefoil from the anterior corneal surface, and it had a smaller effect on the aberrations from the posterior corneal surface. Therefore, the biomechanical effects increased residual wavefront aberrations after SMILE refractive surgery.

Wave front aberrations induced from biomechanical effects after customized myopic laser refractive surgery in finite element model.

PURPOSE: A customized myopic refractive surgery was simulated by establishing a finite element model of the human eye, after which we studied the wave front aberrations induced by biomechanical effects and ablation profile after wave front-guided LASIK surgery. METHODS: Thirty myopia patients (i.e., 60 eyes) without other eye diseases were selected. Their ages, preoperative spherical equivalent, astigmatism, and wave front aberration were then obtained, in addition to the mean spherical equivalent error range - 4 to - 8D. Afterward, wave front-guided customized LASIK surgery was simulated by establishing a finite element eye model, followed by the analysis of the wave front aberrations induced by the surface displacement from corneal biomechanical effects, as well as customized ablation profile. Finally, the preoperative and induced aberrations were statistically analyzed. RESULTS: Comatic aberrations were the main wave front abnormality induced by biomechanical effects, and the wave front aberrations induced by the ablation profile mainly included coma and secondary coma, as well as sphere and secondary-sphere aberrations. Overall, the total high-order aberrations (tHOAs), total coma (C31), and sphere ([Formula: see text]) increased after wave front-guided customized LASIK surgery. According to our correlation analyses, coma, sphere, and tHOAs were significantly correlated with decentration. Additionally, the material parameters of ocular tissue were found to affect the postoperative wave front aberrations. When the material parameters of the sclera remained constant but those of cornea increased, the induced wave front aberrations were reduced. CONCLUSION: All biomechanical effects of cornea and ablation profile had significant effects on postoperative wave front aberrations after customized LASIK refractive surgery; however, the effects of the ablation profile were more notorious. Additionally, the characteristics of biomechanical materials have influence on the clinical correction effect.

Short-Chain Fatty Acids Alleviate Hepatocyte Apoptosis Induced by Gut-Derived Protein-Bound Uremic Toxins.

Chronic kidney disease (CKD) is characterized with the influx of uremic toxins, which impairs the gut microbiome by decreasing beneficial bacteria that produce short-chain fatty acids (SCFAs) and increasing harmful bacteria that produce gut-derived protein-bound uremic toxins (PBUTs). This study aimed to assess the proapoptotic effects of three major gut-derived PBUTs in hepatocytes, and the effects of SCFAs on apoptosis phenotype in vitro. HepG2 (human liver carcinoma cells) and THLE-2 (immortalized human normal liver cells) cell line were incubated with 0, 2, 20, 200, 2000 µM p-cresol sulfate (PCS), indoxyl sulfate (IS), and hippuric acid (HA), respectively, for 24 h. Flow cytometry analysis indicated that three uremic toxins induced varying degrees of apoptosis in hepatocytes and HA represented the highest efficacy. These phenotypes were further confirmed by western blot of apoptosis protein expression [Caspase-3, Caspase-9, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax)]. Human normal hepatocytes (THLE-2) are more sensitive to PBUTs-induced apoptosis compared with human hepatoma cells (HepG2). Mechanistically, extracellular HA could enter hepatocytes, increase reactive oxygen species (ROS) generation, and decrease mitochondrial membrane potential dose-dependently in THLE-2 cells. Notably, coculture with SCFAs (acetate, propionate, butyrate) for 24 h significantly improved HA-induced apoptosis in THLE-2 cells, and propionate (500 µM) represented the highest efficacy. Propionate reduction of apoptosis was associated with improving mitochondria dysfunction and oxidative stress in a manner involving reducing Caspase-3 expression, ROS production, and increasing the Bcl-2/Bax level. As such, our studies validated PBUTs accumulation might be an important cause of liver dysfunction in patients with CKD, and supplementation of SCFAs might be a viable way to protect the liver for patients with CKD.

Biomechanical responses of the cornea after small incision lenticule extraction (SMILE) refractive surgery based on a finite element model of the human eye.

PURPOSE: To investigate the biomechanical responses of the human cornea after small incision lenticule extraction (SMILE) procedures, especially their effects of SMILE surgery on stress and strain. METHODS: Based on finite element analysis, a three-dimensional (3D) model of the human eye was established to simulate SMILE refractive surgery procedures. Stress and strain values were calculated by inputting the intraocular pressure (IOP). RESULTS: After SMILE refractive surgery procedures, the stress and strain of the anterior and posterior corneal surfaces were significantly increased. The equivalent stress and strain on the anterior and posterior corneal surfaces increased with increasing diopter and were concentrated in the central area, whereas the values of stress and strain at the incision site on the anterior surface of the cornea were approximately 0. Compared with the anterior corneal surface, the stress and strain of the posterior surface were larger. Increasing IOP caused an approximately linear change in stress and a nonlinear increase in corneal strain. In addition, we found that the incision sizes and direction had less of an influence on stress and strain. In summary, SMILE surgery increased the equivalent stress and strain on the human cornea. CONCLUSIONS: The equivalent stress and strain of the anterior and posterior human corneal surfaces increased after SMILE refractive surgery; these increases were particularly noticeable on the posterior surface of the cornea.

Hypoxia alleviation-triggered enhanced photodynamic therapy in combination with IDO inhibitor for preferable cancer therapy.

Photodynamic therapy (PDT) has attracted growing attention in the field of cancer therapy due to its non-invasive intervention and initiation of antitumor immune responses by use of non-toxic photosensitizers (PS) and topical light irradiation. However, inherent hypoxia and immunosuppression mediated by checkpoints in tumors severally impair the efficacy of PDT and PDT-induced immunity. Herein, a multi-functional nanoplatform is rationally constructed by fluorinated polymer nanoparticle saturated with oxygen in advance, which simultaneously encapsulated PS (Ce6) and an indoleamine 2,3-dioxygenase (IDO) inhibitor (NLG919). In particular, the tumor hypoxic microenvironment is obviously relieved and much more reactive oxygen species (ROS) is generated by fluorinated nanoparticle compared with alkylated polymer nanoparticle as a control in vitro and in vivo, this is mainly because the fluorinated polymers are endowed with high oxygen carrying capacity which also contributed to the relief of hypoxia. Meanwhile, compared to PDT alone, the co-encapsulation of IDO inhibitor and PS can further greatly enhance efficacy for inhibiting the growth of primary and abscopal tumors via enhanced T cell infiltration. This study can provide a convenient and practical strategy for enhancing the therapeutic effect of PDT and relieving immune suppression, in turn affording clinical benefits for cancer treatment.

A novel design of a polynuclear co-delivery system for safe and efficient cancer therapy.

We report a novel and easy-to-fabricate polynuclear nanoparticle based on the collaborative re-assembly of nanoparticles as a robust chemogene co-delivery platform. Specifically, the polynuclear nanoparticle carrying DOX and siBcl-2 exerts remarkable co-delivery efficiency, increases tumour cell apoptosis and inhibits tumour cell proliferation in vitro and in vivo.

Biocompatible fluorinated poly(ß-amino ester)s for safe and efficient gene therapy.

Cationic polymers have been widely used as one of the most promising non-viral vehicles for gene delivery due to their potential safety and ease of large-scale production. Here, we report the design and synthesis of a series of novel biodegradable fluorinated poly(ß-amino ester)s (FPBAEs) by simple Michael-addition reaction as safe and efficient gene carrier. The results of transfection efficacy assay demonstrated the optimal FPBAE could mediated much higher GFP expression than the commercial transfection agents, polyethyleneimine (PEI, Mw = 25K) and Lipo 2000, as well as the non-fluorinated poly(ß-amino ester)s (PBAE) on both HeLa and HEK-293T cell lines (higher than 70 and 90%, respectively), which was largely attributed to fluorination. Moreover, MTT and hemolysis assay indicated a preferable biocompatibility of FPBAE compared with PEI 25K owing to the low molecular weight and the presence of cleavable ester bonds. Taken together, the novel polymer FPBAE with both excellent gene transfection efficacy and much lower toxicity could serve as a desirable gene vector.

One-step assembly of polymeric demethylcantharate prodrug/Akt1 shRNA complexes for enhanced cancer therapy.

This report demonstrated a one-step assembly for co-delivering chemotherapeutics and therapeutic nucleic acids, constructed by integrating drug molecules into a nucleic acid condensing polymeric prodrug through degradable linkages. Demethylcantharate was selected as the model drug and pre-modified by esterifying its two carboxylic groups with 2-hydroxyethyl acrylate. The synthesized demethylcantharate diacrylate was then used to polymerize with linear polyethyleneimine (PEI 423) through a one-step Michael-addition reaction. The obtained cationic polymeric demethylcantharate prodrug was used to pack Akt1 shRNA into complexes through a one-step assembly. The formed complexes could release the parent drug demethylcantharate and Akt1 shRNA through the hydrolysis of ester bonds. Cellular assays involving cell uptake, cytotoxicity, and cell migration indicated that demethylcantharate and Akt1 shRNA co-delivered in the present form significantly and synergistically suppress the growth and metastasis of three human cancer cells. This work suggests that incorporating drug molecules into a nucleic acid-packing cationic polymer as a polymeric prodrug in a degradable form is a highly convenient and efficient way to co-deliver drugs and nucleic acids for cancer therapy.