A codelivery system loaded with PDL1-siRNA and sorafenib enhances the therapeutic effect of sorafenib on hepatocellular carcinoma via TAT-poly-SS-lysine modified chitosan.

Sorafenib (SF) is a first-line drug for the treatment of hepatocellular carcinoma (HCC) in clinical practice. However, acquired drug resistance tremendously limits the clinical efficacy of sorafenib in treating HCC, which has attracted great attention. PDL1 plays a crucial role in the drug resistance of HCC. Here, a codelivery system based on poly-SS-lysine modified chitosan (TAT-C-SS-P) was established and was applied to deliver sorafenib and PDL1-siRNA for synergetic HCC therapy. The successful synthesis of TAT-C-SS-P was confirmed by 1H NMR. Additionally, sorafenib and PDL1-siRNA were successfully transported into the cells as the decreased expression of VEGF and PD-L1 by administrated with TAT-C-SS-P@SF@ PDL1-siRNA. Simultaneously, the expression of pro-apoptosis proteins cyt-c and Bax was prominently augmented, whereas the expression of anti-apoptosis protein Bcl-2 was decreased. The reduced expression of PDL1 resulted in the downregulation of P-GP and MRP1, which contributed to more sorafenib aggregation in tumor cells. Moreover, TAT-C-SS-P@PDL1-siRNA@SF efficiently promotes apoptosis of HepG2-SI cells, as the apoptosis rate rised to 73 %. A sorafenib-insensitive model was established to evaluate in vivo antitumor effect of TAT-C-SS-P@PDL1-siRNA@SF. TAT-C-SS-P@PDL1-siRNA@SF showed a tumor inhibition rate of 90.2 ± 3.5 % and no significant decrease in body weight. Taken together, our study provided compelling evidence that TAT-C-SS-P@PDL1-siRNA@SF has great potential application in the treatment of HCC clinically.

Catalpol induces apoptosis in breast cancer in vitro and in vivo: Involvement of mitochondria apoptosis pathway and post-translational modifications.

Breast cancer is a fatal cancer with the highest mortality in female. New strategies for anti-breast cancer are still urgently needed. Catalpol, an iridoid glycoside extracted from the traditional Chinese medicinal plant Rehmannia glutinosa, has shown anticancer efficacy in various cancer cells. However, its effect on breast cancer remains unclear. In this study, we aim to investigate the anti-breast cancer activity of catalpol and elucidate its underlying mechanism. Cell counting kit-8 (CCK-8) and morphology change showed that catalpol could inhibit the proliferation and viability of MCF-7 cells. Catalpol administration reduced the tumor volume in xenograft model. Catalpol induced apoptosis in MCF-7 cells confirmed by Hoechst 33342 staining and Annexin V-FITC/PI double staining. In vivo, catalpol also induced apoptosis as seen from the increased level of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) in tumor. According to JC-1 and Dichlorodi-hydrofluorescein Diacetate (DCFH-DA) staining, loss of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation was found in MCF-7 cells treated with catalpol. Furthermore, catalpol also increased the level of cytoplasmic cytochrome c and activity of caspase-3 in MCF-7 cells. Likewise, histopathological and immunohistochemical (IHC) assay also found that catalpol enhanced the levels of cytochrome c and caspase-3 in breast cancer tissues. Ultimately, acetylation, 2-hydroxyisobutyrylation and lactylation were dramatically increased, whereas succinylation, malonylation and phosphorylation were markedly decreased in the breast cancer tumor treated with catalpol. Taken together, catalpol inhibited breast cancer in vitro and in vivo through induction of apoptosis via mitochondria apoptosis pathway and regulation of protein post-translational modifications (PTMs). Thus, it can be considered as an excellent candidate compound for treatment of breast cancer.

Catalpol Ameliorates Neurotoxicity in N2a/APP695swe Cells and APP/PS1 Transgenic Mice.

Alzheimer's disease (AD) causes progressive decline of memory and cognitive deficits. Because of its complicated pathogenesis, the prevention and therapy of AD remain an enormous challenge. It has been reported that catalpol possessed neuroprotective effects against AD. However, the involved mechanism still needs to be intensively studied. Therefore, the effects of catalpol on N2a/APP695swe cells and APP/PS1 mice were identified in the current study. Catalpol could improve cytotoxicity according to CCK-8 assay and ameliorate cellular morphological changes in N2a/APP695swe cells. Neuronal structural damage in the hippocampal CA1 region of APP/PS1 AD mice was improved according to HE staining and immunohistochemistry of NeuN. Meanwhile, catalpol administration ameliorated cognitive deficits confirmed by behavior performance of APP/PS1 mice. Hoechst 33,342 staining and Annexin V-FITC/PI double staining demonstrated that catalpol could reduce apoptosis in N2a/APP695swe cells. Likewise, TUNEL staining also manifested that catalpol significantly reduced apoptosis in hippocampal CA1 region of APP/PS1 mice. Catalpol administration also could improve mitochondrial functions indicated by the ameliorative mitochondrial morphology, the decreased ROS generation, and the increased MMP in N2a/APP695swe cells. Subsequently, catalpol restrained oligomerization of Aß1-42, verified by a reduced ThT fluorescence dose- and time-dependently. Additionally, both Aß1-40 and Aß1-42 aggregation were decreased in N2a/APP695swe cells and APP/PS1 mice administrated with catalpol confirmed by ELISA and western blot. Western blot also showed that catalpol facilitated the phosphorylation of AKT and GSK3ß, and impeded the expression of BACE1 both in vivo and in vitro. Finally, a slight alteration in lactylation, 2-hydroxyisobutyrylation, and phosphorylation were found in N2a/APP695swe cells treated with catalpol. Together, these findings manifested that catalpol served a neuroprotective effect in AD and might be a novel and expecting prophylactic or curative candidate for AD or neurodegenerative diseases therapy.

Saikosaponin-D Mitigates Oxidation in SH-SY5Y Cells Stimulated by Glutamate Through Activation of Nrf2 Pathway: Involvement of PI3K.

Alzheimer's disease (AD) is a typical neurodegenerative disease. Well-established studies have shown an elevated level of ROS (reactive oxygen species) that induces oxidative stress in AD. Saikosaponin-D exhibited significant therapeutic effects on neurodegenerative diseases. However, its in-depth molecular mechanisms against neurotoxicity remain not fully uncovered. Herein, the possible protective effects of saikosaponin-D on glutamate-induced neurotoxicity in SH-SY5Y cells and the underlying mechanism were elucidated. Saikosaponin-D pretreatment could ameliorate glutamate-induced cytotoxicity according to MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and depress apoptosis according to Hoechst 33,342 staining and Annexin V-FITC/PI double staining in SH-SY5Y cells. Additionally, saikosaponin-D administration suppressed oxidative stress in response to glutamate indicated by diminished intracellular ROS formation and reduced MDA (malondialdehyde) content in SH-SY5Y cells. These phenomena, appeared to correlate with the recovered cellular antioxidant enzyme activities and inducted HO-1 (heme oxygenase-1) expression accompanying the nuclear translocation of Nrf2 conduct by saikosaponin-D preconditioning which had been altered by glutamate, were correlated with its neuroprotective. Furthermore, addition of LY294002, a selective inhibitor of PI3K (phosphatidylinositol 3 kinase), blocked saikosaponin-D-caused Nrf2 nuclear translocation and reversed the protection of saikosaponin-D against glutamate in SH-SY5Y cells. Moreover, saikosaponin-D exhibited antioxidant potential with high free radical-scavenging activity as confirmed by a DPPH (2,2-diphenyl-1-picrylhydrazyl) and TEAC (Trolox equivalent antioxidant capacity) in a cell-free system in vitro. Taken together, our results indicated that saikosaponin-D enhanced cellular antioxidant capacity through not only intrinsic free radical-scavenging activity but also induction of endogenous antioxidant enzyme activities and HO-1 expression mediated, at least in part, by activating PI3K and subsequently Nrf2 nuclear translocation, thereby protecting the SH-SY5Y cells from glutamate-induced oxidative cytotoxicity. In concert, these data raise the possibility that saikosaponin-D may be an attractive candidate for prevention and treatment of AD and other diseases related to oxidation in the future.

High-fidelity reprogramming into Leydig-like cells by CRISPR activation and paracrine factors.

Androgen deficiency is a common medical conditions that affects males of all ages. Transplantation of testosterone-producing cells is a promising treatment for male hypogonadism. However, getting a cell source with the characteristics of Leydig cells (LCs) is still a challenge. Here, a high-efficiency reprogramming of skin-derived fibroblasts into functional Leydig-like cells (LLCs) based on epigenetic mechanism was described. By performing an integrated analysis of genome-wide DNA methylation and transcriptome profiling in LCs and fibroblasts, the potentially epigenetic-regulating steroidogenic genes and signaling pathways were identified. Then by using CRISPR/dCas9 activation system and signaling pathway regulators, the male- or female-derived fibroblasts were reprogrammed into LLCs with main LC-specific traits. Transcriptomic analysis further indicated that the correlation coefficients of global genes and transcription factors between LLCs and LCs were higher than 0.81 and 0.96, respectively. After transplantation in the testes of hypogonadal rodent models, LLCs increased serum testosterone concentration significantly. In type 2 diabetic rats model, LLCs which were transplanted in armpit, have the capability to restore the serum testosterone level and improve the hyperglycemia status. In conclusion, our approach enables skin-derived fibroblasts reprogramming into LLCs with high fidelity, providing a potential cell source for the therapeutics of male hypogonadism and metabolic-related comorbidities.

Saikosaponin-A induces apoptosis of cervical cancer through mitochondria- and endoplasmic reticulum stress-dependent pathway in vitro and in vivo: involvement of PI3K/AKT signaling pathway.

Cervical cancer causes considerable mortality in women worldwide. Saikosaponin-A, a triterpenoid glycoside isolated from Bupleurum falcatum, has been proven to exert anti-cancer property. In this study, we evaluated the possibility of saikosaponin-A on cervical cancer in vitro and in vivo. The results showed that saikosaponin-A induced cell death and altered cellular morphology dose-dependently. Saikosaponin-A significantly induced apoptosis in HeLa cells, confirmed by Hoechst 33,342 staining and flow cytometry. Sequentially, saikosaponin-A triggered the mitochondrial-mediated apoptosis demonstrated by deficiency of MMP, induction of Bax/Bcl-2 ratio, leakage of cytochrome c to cytoplasm, and activation of caspase-3. Moreover, ER stress also participated in the apoptosis induced by saikosaponin-A in HeLa cells as indicated by the upregulation of GPR78, CHOP and caspase-12 expression. Furthermore, HeLa cells showed increased expressions of p-PI3K and p-AKT in response to saikosaponin-A treatment. Additionally, saikosaponin-A could inhibit HeLa tumor growth in nude mice and induce apoptosis, reflected by the induction of TUNEL and the expression of cytochrome c, caspase-3 and CHOP confirmed by immunohistochemistry. These findings at least to a certain extent suggested that saikosaponin-A triggered apoptosis through both mitochondrial pathway and ER stress pathway and inhibiting PI3K/Akt signaling, thereby contributing to against cervical cancer. This work provides a new understanding of saikosaponin-A on therapeutic application in treatment of cancer, which has the potential to be a promising candidate therapeutic agent for cervical cancer patients.

Protective effects of crocin against endogenous Aß-induced neurotoxicity in N2a/APP695swe cells.

Alzheimer's disease (AD) is a most common neurodegenerative disorder worldwide. Because of its complex pathogenesis, the prevention and therapies of AD still are a severe challenge. Evidence suggested that crocin, the major component of saffron, exhibited neuroprotective effects in AD. As such, in this study, N2a/APP695swe cells were enrolled to investigate the effects of crocin on endogenous Aß-induced neurotoxicity. Crocin (100 and 200 µM) could ameliorate cytotoxicity according to CCK-8 assay and reduce apoptosis in line with Hoechst 33,342 staining and Annexin V-FITC/PI double staining in N2a/APP695swe cells. Reduced ROS generation and elevated MMP were found in N2a/APP695swe cells treated with crocin (100 and 200 µM). Additionally, crocin at concentrations of 100 and 200 µM inhibited the release of cytochrome and attenuated caspases-3 activity in N2a/APP695swe cells. Furthermore, succinylation, crotonylation, 2-hydroxyisobutyrylation, malonylation, and phosphorylation were significantly reduced, while a slight increase of acetylation was found in 100-µM crocin treated N2a/APP695swe cells. Taken together, crocin may be a promising natural product candidate for the effective cure of AD.

Assessing drug cue-induced brain response in heroin dependents treated by methadone maintenance and protracted abstinence measures.

Methadone maintenance treatment (MMT) and protracted abstinence (PA) are common methods of therapy in heroin addiction as both suppress the craving for drug use. However, the difference in patterns of brain function between two groups is unknown. Functional magnetic resonance imaging (fMRI) based drug cue-reactivity task is a good tool to understand the change of brain function during a certain period of treatment. Twenty-three heroin-dependent patients during PA, 18 heroin-dependent patients during MMT and 20 healthy control (HC) individuals were included to conduct the heroin cue-reactivity task during fMRI. The MMT and PA patients' subjective craving for heroin was evaluated. Differences among the three groups were analyzed with respect to heroin cue induced brain responses. Compared with HC group, MMT and PA groups commonly demonstrated significantly higher brain responses during exposure of heroin-related cues in the bilateral caudate, right thalamus, left hippocampus, parahippocampus, midbrain, left superior parietal lobule, right middle occipital gyrus, left posterior cingulate cortex and right cerebellum. However, compared with PA group, MMT group demonstrated significantly greater brain response mainly in right caudate, hippocampus, midbrain left fusiform, right inferior parietal lobule, left posterior cingulate cortex, cerebellum and postcentral gyrus. No difference in cue induced craving between MMT and PA groups was found. The findings suggest that MMT group demonstrated more enhanced drug cue induced brain responses than PA group, indicating that, these two treatments have different effect on patterns of brain response to heroin related cues in heroin-dependent individuals.

The polymorphism of dopamine D2 receptor TaqIA gene is associated with brain response to drug cues in male heroin-dependent individuals during methadone maintenance treatment.

BACKGROUND: Polymorphism of the dopamine D2 receptor TaqIA gene is related to reward response, relapse risks and effect of therapy for drug addiction. Whether the cue-induced craving and brain response was related to dopamine D2 receptor TaqIA gene is unknown. METHODS: Forty-nine male heroin-dependent individuals [31 with A1 allele of the TaqIA (A1+), 18 A2 allele carriers (A1-)] under methadone maintenance treatment and 20 healthy control subjects performed a heroin cue-reactivity task during functional magnetic resonance imaging. Cue-elicited craving was measured. Difference in cue induced craving and brain response were analyzed among the three groups. Correlation analyses between craving and differential brain response, heroin use and treatment history were performed within A1+ and A1- group respectively. RESULTS: Compared with A1- group, A1+ group showed greater cue-induced response in the ventrolateral prefrontal cortex, medial orbitofrontal gyrus, dorsomedial prefrontal cortex, pallidum, putamen, thalamus, superior parietal lobule and superior occipital gyrus. No difference in craving was found. The response in right thalamus positively correlated with daily heroin and methadone dose in A1+ group. For A1- group, response in left ventral orbitofrontal cortex, medial orbitofrontal gyrus, ventral anterior cingulate cortex, caudate, precuneus, calcarine and bilateral pallidum negatively correlated with duration of heroin use. The response in left ventral orbitofrontal cortex, medial orbitofrontal gyrus, bilateral calcarine and right cerebellum negatively correlated with duration of methadone maintenance treatment in A1- group. CONCLUSIONS: The findings supported that A1 allele of the TaqIA is associated with higher salience allocation to heroin-related cues in heroin-dependent patients.

Longitudinal behavioral and fMRI-based assessment of inhibitory control in heroin addicts on methadone maintenance treatment.

The objective of the present study was to determine whether methadone maintenance treatment (MMT) in heroin-dependent patients affects inhibitory control, whether any MMT-induced changes correlate with methadone dose and MMT duration, and whether these changes depend on the psychological characteristics of patients, such as depression, anxiety and impulsivity. Response inhibition in the GO/NO-GO test was combined with functional magnetic resonance imaging (fMRI) scanning data to examine whether MMT affects inhibitory control in 21 heroin-addicted patients who had already undergone at least three months of MMT. Patients were evaluated one year prior to and after the MMT period. Participants exhibited no difference in their GO/NO-GO reaction time and accuracy rate, or in their false alarm rate under NO-GO conditions. However, increased activation was detected in numerous brain regions in their 12-month fMRI scans, although these were not in the frontal-striatal loop. Increased fMRI activation in the left precentral gyrus and superior temporal gyrus were negatively correlated with the daily methadone dose and total methadone dose during the one-year study period. In conclusion, these results suggested that MMT over one year does not significantly change the behavioral indicators of inhibitory control function in heroin-dependent patients. The increase in activation leads to the hypothesis that MMT over one year may increase cognitive inhibitory control, which may be the result of the combined negative effect of methadone and the positive effect of functional recovery after withdrawal of heroin.

Disrupted coupling of large-scale networks is associated with relapse behaviour in heroin-dependent men.

BACKGROUND: It is unknown whether impaired coupling among 3 core large-scale brain networks (salience [SN], default mode [DMN] and executive control networks [ECN]) is associated with relapse behaviour in treated heroin-dependent patients. METHODS: We conducted a prospective resting-state functional MRI study comparing the functional connectivity strength among healthy controls and heroin-dependent men who had either relapsed or were in early remission. Men were considered to be either relapsed or in early remission based on urine drug screens during a 3-month follow-up period. We also examined how the coupling of large-scale networks correlated with relapse behaviour among heroin-dependent men. RESULTS: We included 20 controls and 50 heroin-dependent men (26 relapsed and 24 early remission) in our analyses. The relapsed men showed greater connectivity than the early remission and control groups between the dorsal anterior cingulate cortex (key node of the SN) and the dorsomedial prefrontal cortex (included in the DMN). The relapsed men and controls showed lower connectivity than the early remission group between the left dorsolateral prefrontal cortex (key node of the left ECN) and the dorsomedial prefrontal cortex. The percentage of positive urine drug screens positively correlated with the coupling between the dorsal anterior cingulate cortex and dorsomedial prefrontal cortex, but negatively correlated with the coupling between the left dorsolateral prefrontal cortex and dorsomedial prefrontal cortex. LIMITATIONS: We examined deficits in only 3 core networks leading to relapse behaviour. Other networks may also contribute to relapse. CONCLUSION: Greater coupling between the SN and DMN and lower coupling between the left ECN and DMN is associated with relapse behaviour. These findings may shed light on the development of new treatments for heroin addiction.

Regional homogeneity changes between heroin relapse and non-relapse patients under methadone maintenance treatment: a resting-state fMRI study.

BACKGROUND: Methadone maintenance treatment (MMT) is recognized as one of the most effective treatments for heroin addiction but its effect is dimmed by the high incidence of heroin relapse. However, underlying neurobiology mechanism of heroin relapse under MMT is still largely unknown. Here, we took advantage of a resting-state fMRI technique by analysis of regional homogeneity (ReHo), and tried to explore the difference of brain function between heroin relapsers and non-relapsers in MMT. METHODS: Forty MMT patients were included and received a 12-month follow-up. All patients were given baseline resting-state fMRI scans by using a 3.0 T GE Signa Excite HD whole-body MRI system. Monthly self-report and urine test were used to assess heroin relapse or non-relapse. Subjective craving was measured with visual analog scale. The correlation between ReHo and the degree of heroin relapse was analyzed. RESULTS: Compared with the non-relapsers, ReHo values were increased in the bilateral medial orbitofrontal cortex, right caudate, and right cerebellum of the heroin relapsers while those in the left parahippocampal gyrus, left middle temporal gyrus, right lingual gyrus, and precuneus were decreased in heroin relapsers. Importantly, altered ReHo in the right caudate were positively correlated with heroin relapse rates or subjective craving response. CONCLUSIONS: Using the resting-state fMRI technique by analysis of ReHo, we provided the first resting-state fMRI evidence that right caudate may serve as a potential biomarker for heroin relapse prediction and also as a promising target for reducing relapse risk.

Calcitonin gene-related peptide promotes the expression of osteoblastic genes and activates the WNT signal transduction pathway in bone marrow stromal stem cells.

Calcitonin gene-related peptide (CGRP) is known to induce osteoblastic differentiation and alkaline phosphatase activity in bone marrow stromal stem cells (BMSCs). However, it has remained elusive whether this effect is mediated by CGRP receptors directly or whether other signaling pathways are involved. The present study assessed the possible involvement of the Wnt/ß­catenin signaling pathway in the activation of CGRP signaling during the differentiation of BMSCs. First, the differentiation of BMSCs was induced in vitro and the expression of CGRP receptors was examined by western blot analysis. The effects of exogenous CGRP and LiCl, a stimulator of the Wnt/ß­catenin signaling pathway, on the osteoblastic differentiation of BMSCs were assessed; furthermore, the expression of mRNA and proteins involved in the Wnt/ß­catenin signaling pathway was assessed using quantitative PCR and western blot analyses. The results revealed that CGRP receptors were expressed throughout the differentiation of BMSCs, at days 7 and 14. Incubation with CGRP and LiCl led to the upregulation of the expression of osteoblastic genes associated with the Wnt/ß­catenin pathway, including the mRNA of c­myc, cyclin D1, Lef1, Tcf7 and ß­catenin as well as ß­catenin protein. However, the upregulation of these genes and ß­catenin protein was inhibited by CGRP receptor antagonist or secreted frizzled­related protein, an antagonist of the Wnt/ß­catenin pathway. The results of the present study therefore suggested that the Wnt/ß-catenin signaling pathway may be involved in CGRP­ and LiCl-promoted osteoblastic differentiation of BMSCs.

Brain white matter integrity in heroin addicts during methadone maintenance treatment is related to relapse propensity.

INTRODUCTION: Cognitive deficits caused by heroin-induced white matter (WM) impairments hinder addicts' engagement in and benefit from treatment. The predictive value of WM integrity in heroin addicts during methadone maintenance treatment (MMT) for future relapse is unclear. METHODS: Forty-eight MMT patients were given baseline diffusion tensor imaging scans and divided into heroin relapsers (HR, 25 cases) and abstainers (HA, 23 cases) according to the results of 6-month follow-up. Intergroup comparisons were performed for fractional anisotropy (FA), radial diffusivity (RD), and axial diffusivity (AD). The correlation between diffusion tensor imaging indices and the degree of heroin relapse was analyzed. RESULTS: Compared with HA group, HR group had reduced FA in the right retrolenticular part, left anterior and posterior limb of internal capsule, bilateral anterior corona radiata, and right external capsule. Three out of the six regions showed increased RD, with no changes in AD. The FA and AD values in the left posterior limb of internal capsule correlated negatively with the heroin-positive urinalysis rate within follow-up. CONCLUSIONS: Lower WM integrity in MMT patients may add to neurobiological factors associated with relapse to heroin use. Strategies for improving WM integrity provide a new perspective to prevent future relapse to heroin abuse.

Methadone-induced Damage to White Matter Integrity in Methadone Maintenance Patients: A Longitudinal Self-control DTI Study.

Methadone maintenance treatment (MMT) can induce impairments in brain function and structure, despite its clinical effectiveness. However, the effect of chronic MMT on brain white matter (WM) is not fully known. Thirty-three MMT patients underwent diffusion tensor imaging (DTI) twice - at the start of the study (Scan1) and one year later (Scan2). Tract-based spatial statistics were used to investigate changes in fractional anisotropy (FA), axial diffusivity (AD) and radial diffusivity (RD) between the two scans. The correlations between DTI indices and methadone consumption and neuropsychological status were analysed. We found significantly decreased FA, decreased AD and increased RD in Scan2 in extensive WM regions; overlapping regions were found in the left posterior limb and the retrolenticular part of internal capsule, superior and posterior corona radiata, bilateral external capsule and the right superior longitudinal fasciculus. In addition, the change of FA in the overlapping regions was positively correlated with the accumulated dosage of methadone use, the RD value in Scan2 and non-planning impulsiveness (NPI) measured at follow-up. The results suggest that methadone has damaging effects on WM integrity. The dose-dependent pattern and characteristics of the impairment may suggest new strategies for MMT.