Inhibition of GSDMD-dependent pyroptosis decreased methamphetamine self-administration in rats.

It is widely believed that the activation of the central dopamine (DA) system is crucial to the rewarding effects of methamphetamine (METH) and to the behavioral outcomes of METH use disorder. It was reported that METH exposure induced gasdermin D (GSDMD)-dependent pyroptosis in rats. The membrane pore formation caused by METH-induced pyroptosis may also contribute to the overflow of DA into the extracellular space and subsequently increase the DA levels in the brain. The present study firstly investigated whether the membrane pore information induced by GSDMD-dependent pyroptosis was associated with the increased DA levels in the ventral tegmental area (VAT) and nucleus accumbens (NAc) of rats self-administering METH and SY-SH5Y cells treated by METH. Subsequently, the effect of pore formation blockade or genetic inhibition of GSDMD on the reinforcing and motivational effect of METH was determined in rats, using the animal model of METH self-administration (SA). METH exposure significantly increased the activity of NLRP1/Cas-1/GSDMD pathway and the presence of pyroptosis, accompanied by the significantly increased DA levels in VTA and NAc. Moreover, intraperitoneal injections of disulfiram (DSF) or microinjection of rAAV-shGSDMD into VTA/NAc significantly reduced the reinforcing and motivational effect of METH, accompanied by the decreased level of DA in VTA and NAc. The results provided novel evidence that METH-induced pyroptosis could increase DA release in VTA and NAc via the NLRP1/Cas-1/GSDMD pathway. Additionally, membrane pores or GSDMD blockade could significantly reduce the reinforcing and motivational effect of METH. In conclusion, blocking GSDMD and membrane pore formation could be a promising potential target for the development of agents to treat METH use disorder.

USP33 enhances cell survival and stemness by deubiquitinating CTNNB1 in BXPC-3 and SW1990 cells.

Ubiquitin-specific protease 33 (USP33) has been implicated in various cancers, but its biological function and mechanism of action remain unknown in pancreatic cancer (PCa) as a deubiquitinating enzyme. Herein, we report that USP33 silencing inhibits PCa cell survival and self-renewal. USPs highly expressed in spherical PCa cells were screened by comparing the levels of ubiquitin-specific proteases in spherical PCa cells and adherent PCa cells. After silencing USP, the effect of USP on the proliferation of PCa cells was detected by CCK-8 and colony formation assay, and the effect of USP on cell stemness was detected by tumor sphere formation assay, flow analysis, and western blot analysis. The interaction of USP with CTNNB1 and the effect of USP on the ubiquitination of CTNNB1 were verified by coimmunoprecipitation assay. After replenishing CTNNB1, cell proliferation and cell stemness were examined. USP33 is upregulated in spheric BXPC-3, PCNA-1, and SW1990, compared with adherent BXPC-3, PCNA-1, and SW1990. USP33 interacts with CTNNB1, and stabilizes CTNNB1 by suppressing its degradation. Furthermore, cell proliferation, colony-forming, and self-renewal abilities of PCa cells in vitro, and the expression of stem cell markers EpCAM and CD44, C-myc, Nanog, and SOX2, were suppressed when USP33 was knocked down, which was reversed when CTNNB1 was ectopically expressed in PCa cells. Thus, USP33 promotes PCa cell proliferation and self-renewal by inhibiting the degradation of CTNNB1. USP33 inhibition may be a new treatment option for PCa patients.

EGCG attenuates METH self-administration and reinstatement of METH seeking in mice.

Methamphetamine (METH) use disorder is a chronic, relapsing disorder and involves frequent failures of self-control of drug seeking and taking. Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenolic compounds of green tea, which has shown great therapeutic effectiveness in neurological disorders. However, it is still unknown whether and how EGCG affects METH seeking behaviour. Here, we show nanostructured EGCG/ascorbic acid nanoparticles (EGCG/AA NPs) dose-dependently reduced METH self-administration (SA) under fixed-ratio 1 (FR1) and progressive ratio (PR) reinforcement schedules in mice and shifted METH dose-response curves downward. Furthermore, EGCG/AA NPs decreased drug- and cue-induced METH seeking. In addition, we found that METH SA led to a decrease in inhibitory postsynaptic currents (IPSCs) and increase in the AMPAR/NMDAR ratio and excitation/inhibition (E/I) ratio in ex vivo midbrain slices from ventral tegmental area (VTA) dopamine neurons. EGCG/AA NPs enhanced Gamma-aminobutyric acid (GABA)ergic inhibition and normalized the E/I ratio. EGCG restored the balance between excitation and inhibition in VTA dopamine neurons, which may contribute to the attenuation of METH SA. These findings indicate that EGCG is a promising pharmacotherapy for METH use disorder.

The guanine nucleotide exchange factor VAV3 participates in ERBB4-mediated cancer cell migration.

ERBB4 is a member of the epidermal growth factor receptor (EGFR)/ERBB subfamily of receptor tyrosine kinases that regulates cellular processes including proliferation, migration, and survival. ERBB4 signaling is involved in embryogenesis and homeostasis of healthy adult tissues, but also in human pathologies such as cancer, neurological disorders, and cardiovascular diseases. Here, an MS-based analysis revealed the Vav guanine nucleotide exchange factor 3 (VAV3), an activator of Rho family GTPases, as a critical ERBB4-interacting protein in breast cancer cells. We confirmed the ERBB4-VAV3 interaction by targeted MS and coimmunoprecipitation experiments and further defined it by demonstrating that kinase activity and Tyr-1022 and Tyr-1162 of ERBB4, as well as the intact phosphotyrosine-interacting SH2 domain of VAV3, are necessary for this interaction. We found that ERBB4 stimulates tyrosine phosphorylation of the VAV3 activation domain, known to be required for guanine nucleotide exchange factor (GEF) activity of VAV proteins. In addition to VAV3, the other members of the VAV family, VAV1 and VAV2, also coprecipitated with ERBB4. Analyses of the effects of overexpression of dominant-negative VAV3 constructs or shRNA-mediated down-regulation of VAV3 expression in breast cancer cells indicated that active VAV3 is involved in ERBB4-stimulated cell migration. These results define the VAV GEFs as effectors of ERBB4 activity in a signaling pathway relevant for cancer cell migration.

All-trans retinoic acid regulating angiopoietins-1 and alleviating extracellular matrix accumulation in interstitial fibrosis rats.

All-trans retinoic acid (ATRA) is one of essentially active metabolite of vitamin A, and plays an important role in diverse physiological processes, such as cellular growth and function. Renal interstitial fibrosis (RIF) is a common pathological characteristic of chronic renal disease causing end-stage renal disease currently lacking effective treatment. Low level of Angiopoietins-1 (Angpt-1) is associated with extracellular matrix accumulation and fibrosis diseases. This study was performed to assess the association of ATRA with Angpt-1 in RIF disease. Rats were divided into three groups: group of sham (SHO group), group of unilateral ureteral obstruction group (UUO group), UUO mice administrated daily at the dose of ATRA (ATRA group). Masson-staining was used to detect the histologic lesion. Immunohistochemistry and Western-blot were applied to determine the targeted proteins. RIF score was significantly increased in UUO rats when compared with that of SHO group, and the fibrosis score was notably reduced in ATRA group. Transforming growth factor-ß1 (TGF-ß1), collagen IV (Col-IV) and fibronectin (FN) expressions in UUO group were significantly up-regulated, whereas Angpt-1 expression was significantly down-regulated compared with the SHO group. ATRA treatment reduced TGF-ß1, Col-IV and FN expressions and improved Angpt-1 expression compared with the UUO group. The protein expression of Angpt-1 in kidney tissue of UUO group was negatively correlated with RIF index and protein expressions of Col-IV, FN and TGF-ß1. In conclusion, low expression of Angpt-1 was associated with the RIF disease and ATRA treatment can increase the Angpt-1 and alleviate the RIF lesion in UUO rats.

Relationship between TGF-ß1 + 869 T/C and + 915 G/C gene polymorphism and risk of acute rejection in renal transplantation recipients.

BACKGROUND: This meta-analysis was conducted to assess the relationship between the transforming growth factor-beta 1 (TGF-ß1) + 869 T/C gene polymorphism, + 915 G/C gene polymorphism, and the susceptibility of acute rejection in the recipients with renal transplantation. METHODS: Relevant studies were searched and identified from the Cochrane Library and PubMed, and eligible investigations were recruited and data were calculated by meta-analysis. RESULTS: In this study, we found no relationship between either TGF-ß1 + 869 T/C or TGF-ß1 + 915 G/C gene polymorphism and acute rejection susceptibility in patients with renal transplantation. No association between either gene polymorphism and acute rejection susceptibility in patients with renal transplantation in Caucasian, Asian, or African populations individually was found. CONCLUSION: The TGF-ß1 + 869 T/C and + 915 G/C gene polymorphisms are not associated with acute rejection susceptibility in recipients with renal transplantation.

Association of hypoxia-inducible factor-1α (HIF1α) 1790G/A gene polymorphism with renal cell carcinoma and prostate cancer susceptibility: a meta-analysis.

BACKGROUND: This meta-analysis was performed to evaluate the relationship between hypoxia-inducible factor-1α (HIF1α) 1790G/A gene polymorphism and the susceptibility to renal cell carcinoma (RCC) and prostate cancer (PCa). METHODS: Association investigations were identified and included from the Embase, Cochrane Library and PubMed databases on March 1, 2018, and eligible investigations were analyzed by meta-analysis. Odds ratios (OR) were used to express the dichotomous data, and the 95% confidence intervals (CI) were also calculated. RESULTS: In this meta-analysis, we found that the AA genotype of HIF1α 1790G/A was positively associated with the risk of RCC in overall populations, Caucasians, but not for Asians. G allele and GG genotype were not associated with the susceptibility of RCC in overall populations, Caucasians, and Asians. The G allele was negatively associated with PCa susceptibility in overall populations, Asians, but not for Caucasians. GG genotype was negatively associated with PCa susceptibility in Asians, but not for overall populations and Caucasians. HIF1α 1790G/A AA genotype was not associated with PCa susceptibility in overall populations of Caucasians or Asians. CONCLUSION: AA genotype of HIF1α 1790G/A was positively associated with RCC risk in overall populations and Caucasians. Furthermore, the G allele was negatively associated with prostate cancer susceptibility in overall populations, Asians, and GG genotype was negatively associated with PCa susceptibility in Asians.

p53-dependent transcription and tumor suppression are not affected in Set7/9-deficient mice.

Methylation of specific lysine residues in the C terminus of p53 is thought to govern p53-dependent transcription following genotoxic and oncogenic stress. In particular, Set7/9 (KMT7)-mediated monomethylation of human p53 at lysine 372 (p53K372me1) was suggested to be essential for p53 activation in human cell lines. This finding was confirmed in a Set7/9 knockout mouse model (Kurash et al., 2008). In an independent knockout mouse strain deficient in Set7/9, we have investigated its involvement in p53 regulation and find that cells from these mice are normal in their ability to induce p53-dependent transcription following genotoxic and oncogenic insults. Most importantly, we detect no impairment in canonical p53 functions in these mice, indicating that Set7/9-mediated methylation of p53 does not seem to represent a major regulatory event and does not appreciably control p53 activity in vivo.

Multitarget Therapy: An Effective and Safe Therapeutic Regimen for Lupus Nephritis.

INTRODUCTION: We evaluated the effectiveness and safety of various multitarget therapies for inducing remission in lupus nephritis patients. METHODS: Randomized controlled trials (RCT) were identified and extracted from the Embase, PubMed, Chinese Biomedical Literature Database (CBM), and the Cochrane Library until Oct 31, 2018, investigations meeting inclusion criteria were extracted, and data were analyzed by meta-analysis. The total remission (TR; complete to partial remission), complete remission (CR), albumin, proteinuria levels, negative rate of anti-double-stranded DNA antibody (ds-DNA), negative rate of anti-nuclear antibody (ANA), and systemic lupus erythematosus disease activity index (SLE-DAI) were calculated using the software of RevMan 5.3. RESULTS: Eleven RCTs were included and analyzed. The multitarget therapy group exhibited a higher value of CR (OR=3.06, 95%CI: 2.35-3.99, P﹤0.00001) as well as TR (OR=3.83, 95%CI: 2.77-5.31, P﹤0.00001) than those in the cyclophosphamide (CYC) group. In addition, multitarget therapies had more albumin (WMD=3.50, 95%CI: 1.04-5.95, P=0.005), greater albumin increases (OR=1.96, 95%CI: 0.63-3.29, P=0.004) and higher negative rates of ds-DNA (OR=2.13, 95%CI: 1.51-3.01, P﹤0.0001) and ANA (OR=2.82, 95%CI: 1.77-4.50, P﹤0.0001) when compared with the CYC group. This group also had less proteinuria levels (WMD=-0.55, 95%CI: -0.79 to -0.30, P﹤0.0001), lower degrees of SLE-DAI (OR=-1.80, 95%CI:-2.78 to -0.81, P=0.0004), and a lower adverse reaction rate. For example, gastrointestinal syndrome, irregular menstruation and leucopenia happened less frequently in the multitarget therapy group. However, hypertension was more prevalent in the multitarget therapy group. CONCLUSIONS: Multitarget therapy is an effective and safe intervention for inducing remission in lupus nephritis patients.

Acupuncture Reduces Apoptosis of Granulosa Cells in Rats with Premature Ovarian Failure Via Restoring the PI3K/Akt Signaling Pathway.

Acupuncture is widely recognized as an effective therapy for premature ovarian failure (POF) in clinical, but information about its potential mechanisms is rarely explored. To investigate the mechanism, fifty SD female rats were randomly divided into normal group, POF group, POF+estradiol-valerate group (abbreviated as estradiol group), and POF+acupuncture group (abbreviated as acupuncture group). The estrous cycle of the rats was tracked by vaginal smears. Their ovaries morphology was observed by hematoxylin-eosin staining. The apoptotic level of granulosa cells was detected by in situ TUNEL fluorescence staining assay. Serum follicle-stimulating hormone (FSH) and estrogen (E2) levels were measured by enzyme-linked-immunosorbent-assay (ELISA). Protein and gene expression of PI3K, Akt, bcl-2, and bax were detected by Western blotting and qPCR. In the acupuncture and estradiol groups, compared with the POF group as controls, the apoptosis number of granulosa cells was significantly decreased (p < 0.05). FSH levels were decreased, while E2 levels were increased (p > 0.05). The gene and protein expression levels of PI3K, Akt, and bcl-2 were increased, while the expression levels of bax were decreased (p < 0.05), and the protein expression level of p-Akt increased. There was no significant difference between the acupuncture group and the estradiol group (p > 0.05). Acupuncture was able to regulate hormone levels in POF rats, up-regulate PI3K/Akt signaling pathway, and reduce the apoptosis of granulosa cells. This may be one of the mechanisms of acupuncture treating premature ovarian failure.

Quantitative Protein Sulfenic Acid Analysis Identifies Platelet Releasate-Induced Activation of Integrin ß2 on Monocytes via NADPH Oxidase.

Physiological stimuli such as thrombin, or pathological stimuli such as lysophosphatidic acid (LPA), activate platelets. The activated platelets bind to monocytes through P-selectin-PSGL-1 interactions but also release the contents of their granules, commonly called "platelet releasate". It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS is considered to be a potential regulator of protein function. In a previous study, we used THP-1 monocytic cells exposed to LPA- or thrombin-induced platelet releasate and a modified biotin switch assay to unravel the biological processes that are influenced by reversible cysteine oxidation. To gain a better understanding of the redox regulation of monocytes in atherosclerosis, we have now altered the modified biotin switch to selectively quantify protein sulfenic acid, a subpopulation of reversible cysteine oxidation. Using arsenite as reducing agent in the modified biotin switch assay, we were able to quantify 1161 proteins, in which more than 100 sulfenic acid sites were identified. Bioinformatics analysis of the quantified sulfenic acid sites highlighted the relevant, previously missed biological process of monocyte transendothelial migration, which included integrin ß2. Flow cytometry validated the activation of LFA-1 (αLß2) and Mac-1 (αMß2), two subfamilies of integrin ß2 complexes, on human primary monocytes following platelet releasate treatment. The activation of LFA-1 was mediated by ROS from NADPH oxidase (NOX) activation. Production of ROS and activation of LFA-1 in human primary monocytes were independent of P-selectin-PSGL-1 interaction. Our results proved the modified biotin switch assay to be a powerful tool with the ability to reveal new regulatory mechanisms and identify new therapeutic targets.

Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

Associations of hypertension, diabetes and heart disease risk with body mass index in older Chinese adults: a population-based cohort study.

OBJECTIVE: Obesity is a well-established risk factor for disease. Controversy exists regarding the relative risk of morbidity and mortality in individuals who are overweight or underweight compared with individuals with a normal body mass index (BMI). In this study, we investigated the associations between BMI and three non-communicable diseases (hypertension, diabetes and heart disease) in older adults. DESIGN: Cohort study. SETTING: This study used data from the China Health and Retirement Longitudinal Study. The baseline survey was carried out in 2011, and follow-up surveys were conducted in 2013, 2015 and 2018. PARTICIPANTS: Participants who reported having no doctor-diagnosed chronic disease at baseline were included in this study. MAIN OUTCOME MEASURES: We analysed the association between baseline BMI and disease incidence using Cox proportional hazards models. Disease information included self-reported diagnosed conditions. BMI was categorised according to the standard Chinese criteria: underweight (<18.5 kg/m2), normal body weight (18.5-23.9 kg/m2), overweight (24.0-27.9 kg/m2) and obese (≥28.0 kg/m2). RESULTS: A total of 5605 participants were included at baseline. Based on the Kaplan-Meier estimation, the participants who were obese had the highest incidence of all three diseases. Compared with normal weight participants, overweight participants had a greater disease incidence (log-rank tests are p<0.01). Cox regression models showed that with increasing BMI, the HRs of diseases increased accordingly (eg, for hypertension, compared with the BMI group <18.5 kg/m2, the HRs for the BMI groups 18.5-23.9, 24.0-27.9 and ≥28.0 were 1.43 (95% CI 1.00 to 2.05), 2.19 (95% CI 1.51 to 3.18) and 2.89 (95% CI 1.91 to 4.36), respectively). CONCLUSION: A higher BMI was associated with an increased risk of hypertension, diabetes and heart disease in the population aged 45 years and older. Even within normal BMI ranges, a higher BMI was associated with an increased risk of disease. Actions are urgently needed at the population level to address the growing public health challenge of excess weight in the context of an ageing population.

1-Norm waveform analysis for MR elastography-based quantification of inhomogeneity: Effects of the freeze-thaw cycle and Alzheimer's disease.

BACKGROUND: Despite its success in the mechanical characterization of biological tissues, magnetic resonance elastography (MRE) uses ill-posed wave inversions to estimate tissue stiffness. 1-Norm has been recently introduced as a mathematical measure for the scattering of mechanical waves due to inhomogeneities based on an analysis of the delineated contours of wave displacement. PURPOSE: To investigate 1-Norm as an MRE-based quantitative biomarker of mechanical inhomogeneities arising from microscopic structural tissue alterations caused by the freeze-thaw cycle (FTC) or Alzheimer's disease (AD). METHODS: In this proof-of-concept study, we prospectively investigated excised porcine kidney (n = 6), liver (n = 6), and muscle (n = 6) before vs. after the FTC at 500-2000 Hz and excised murine brain of healthy controls (n = 3) vs. 5xFAD species with AD (n = 3) at 1200-1800 Hz using 0.5 T tabletop MRE. 1-Norm analysis was compared with conventional wave inversion. RESULTS: While the FTC reduced both stiffness and inhomogeneity in kidney, liver, and muscle tissue, AD led to lower brain stiffness but more pronounced mechanical inhomogeneity. CONCLUSION: Our preliminary results show that 1-Norm is sensitive to tissue mechanical inhomogeneity due to FTC and AD without relying on ill-posed wave inversion techniques. 1-Norm has the potential to be used as an MRE-based diagnostic biomarker independent of stiffness to characterize abnormal conditions that involve changes in tissue mechanical inhomogeneity.

The role of heterodimers formed by histamine H3 receptors and dopamine D1 receptors on the methamphetamine-induced conditioned place preference.

RATIONALE: The rewarding effect of Methamphetamine (METH) is commonly believed to play an important role in METH use disorder. The altered expression of dopamine D1 receptor (D1R) has been suggested to be essential to the rewarding effect of METH. Notably, D1R could interact with histamine H3 receptors (H3R) by forming a H3R-D1R heteromer (H3R-D1R). OBJECTIVES: This study was designed to specifically investigate the involvement of H3R-D1R in the rewarding effect of METH. METHODS: C57BL/6 mice were treated with intraperitoneal injections of a selective H3R antagonist (Thioperamide, THIO; 20 mg/kg), an H1R antagonist (Pyrilamine, PYRI; 10 mg/kg), or microinjections of cytomegalovirus (CMV)-transmembrane domain 5 (TM5) into the nucleus accumbens (NAc). The animal model of Conditioned Place Preference (CPP) was applied to determine the impact of H3R-D1R on the rewarding effect of METH. RESULTS: METH resulted in a significant preference for the drug-associated chamber, in conjunction with increased H3R and decreased D1R expression in both NAc and the ventral tegmental area (VTA). THIO significantly attenuated the rewarding effect of METH, accompanied by decreased H3R and increased D1R expression. In contrast, pyrilamine failed to produce the similar effects. Moreover, the inhibitory effect of THIO on METH-induced CPP was reversed by SKF38393, a D1R agonist. Furthermore, SCH23390, a D1R antagonist, counteracted the ameliorative effect of SKF38393 on THIO. Co-immunoprecipitation (CO-IP) experiments further demonstrated the specific interaction between H3R and D1R in METH CPP mice. The rewarding effect of METH was also significantly blocked by the interruption of CMV-transmembrane domain 5 (TM5), but not CMV-transmembrane domain 7 (TM7) in NAc. CONCLUSION: These results suggest that modulating the activity of H3R-D1R complex holds promise for regulating METH use disorder and serves as a potential drug target for its treatment.

The neuroprotective effect of LCZ696 on methamphetamine-induced cognitive impairment in mice.

OBJECTIVE: Methamphetamine (METH) exposure commonly causes cognitive impairment. An angiotensin II receptor/neprilysin inhibitor (ARNI), LCZ696 has been demonstrated to inhibit inflammation, oxidative stress and apoptosis. The present study was designed to examine the effect of LCZ696 on METH-induced cognitive impairment and the underlying mechanism. METHODS: Following daily treatment of either saline or METH (5 mg/kg) for 5 consecutive days, the cognitive function was tested using the Y-maze and the Novel Object Recognition (NOR) in Experiment 1. In Experiment 2, mice were initially treated with saline or LCZ696 (60 mg/kg) for 9 consecutive days, followed by LCZ696, METH or saline for 5 days. Cognitive testing was carried out as Experiment 1. In Experiment 3, SH-SY5Y cells were treated with either METH (2.5 Mm) or ddH2O for 12 h. The apoptosis and reactive oxygen species (ROS) level of SH-SY5Y were examined. In Experiment 4, SH-SY5Y cells were pretreated with either ddH2O or LCZ696 (70um) for 30 min, followed by ddH2O or METH treatment for 12 h. Nrf2 and HO-1 protein expression was examined in the ventral tegemental area (VTA) of all the animals and SH-SY5Y cells. RESULTS: LCZ696 significantly improved METH-induced cognitive impairment, in conjunction with decreased apoptosis and ROS levels in VTA of METH-treated mice and SH-SY5Y cells. METH significantly decreased Nrf2 and HO-1 protein expression in VTA of mice and SH-SY5Y cells, which was reversed by LCZ696 treatment. CONCLUSION: LCZ696 yields a neuroprotective effect against METH-induced cognitive dysfunction via the Nrf2/HO-1 signaling pathway.

ThermomiR-377-3p-induced suppression of Cirbp expression is required for effective elimination of cancer cells and cancer stem-like cells by hyperthermia.

BACKGROUND: In recent years, the development of adjunctive therapeutic hyperthermia for cancer therapy has received considerable attention. However, the mechanisms underlying hyperthermia resistance are still poorly understood. In this study, we investigated the roles of cold­inducible RNA binding protein (Cirbp) in regulating hyperthermia resistance and underlying mechanisms in nasopharyngeal carcinoma (NPC). METHODS: CCK-8 assay, colony formation assay, tumor sphere formation assay, qRT-PCR, Western blot were employed to examine the effects of hyperthermia (HT), HT + oridonin(Ori) or HT + radiotherapy (RT) on the proliferation and stemness of NPC cells. RNA sequencing was applied to gain differentially expressed genes upon hyperthermia. Gain-of-function and loss-of-function experiments were used to evaluate the effects of RNAi-mediated Cirbp silencing or Cirbp overexpression on the sensitivity or resistance of NPC cells and cancer stem-like cells to hyperthermia by CCK-8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay, and in subcutaneous xenograft animal model. miRNA transient transfection and luciferase reporter assay were used to demonstrate that Cirbp is a direct target of miR-377-3p. The phosphorylation levels of key members in ATM-Chk2 and ATR-Chk1 pathways were detected by Western blot. RESULTS: Our results firstly revealed that hyperthermia significantly attenuated the stemness of NPC cells, while combination treatment of hyperthermia and oridonin dramatically increased the killing effect on NPC cells and cancer stem cell (CSC)­like population. Moreover, hyperthermia substantially improved the sensitivity of radiation­resistant NPC cells and CSC­like cells to radiotherapy. Hyperthermia noticeably suppressed Cirbp expression in NPC cells and xenograft tumor tissues. Furthermore, Cirbp inhibition remarkably boosted anti­tumor­killing activity of hyperthermia against NPC cells and CSC­like cells, whereas ectopic expression of Cirbp compromised tumor­killing effect of hyperthermia on these cells, indicating that Cirbp overexpression induces hyperthermia resistance. ThermomiR-377-3p improved the sensitivity of NPC cells and CSC­like cells to hyperthermia in vitro by directly suppressing Cirbp expression. More importantly, our results displayed the significantly boosted sensitization of tumor xenografts to hyperthermia by Cirbp silencing in vivo, but ectopic expression of Cirbp almost completely counteracted hyperthermia-mediated tumor cell-killing effect against tumor xenografts in vivo. Mechanistically, Cirbp silencing-induced inhibition of DNA damage repair by inactivating ATM-Chk2 and ATR-Chk1 pathways, decrease in stemness and increase in cell death contributed to hyperthermic sensitization; conversely, Cirbp overexpression-induced promotion of DNA damage repair, increase in stemness and decrease in cell apoptosis contributed to hyperthermia resistance. CONCLUSION: Taken together, these findings reveal a previously unrecognized role for Cirbp in positively regulating hyperthermia resistance and suggest that thermomiR-377-3p and its target gene Cirbp represent promising targets for therapeutic hyperthermia.

The Expression and Molecular Mechanisms of Matrix Metalloproteinase-9 and Vascular Endothelial Growth Factor in Renal Interstitial Fibrosis in Rats.

To explore a new approach for the treatment of renal interstitial fibrosis (RIF), we detected the expression of matrix metalloproteinase-9 (MMP9) and vascular endothelial growth factor (VEGF). Twenty-four male Sprague Dawley (SD) rats were randomly divided into 2-week normal control (2NC) group, 4-week NC (4NC) group, 2- week unilateral ureteral obstruction (2UUO) group, and 4-week UUO (4UUO) group. We performed left ureteral ligation on UUO groups. Then, we sacrificed the rats of the 2NC group and 2UUO group at 2 weeks and the other groups at 4 weeks after the surgery. Immunohistochemistry and western blot were applied to detect the expression of MMP9, VEGF, fibronectin (FN), type IV collagen (Col-IV), and transforming growth factor-ß1 (TGF-ß1). MMP9 levels reduced after UUO surgery. Its expression was less in the 4UUO group than in the 2UUO group (P<0.05). The expression of VEGF, TGF- ß1, FN, and Col-IV was higher in UUO groups than in NC groups (P<0.05). The expression of these indicators was higher in the 4UUO group than in the 2UUO group (P<0.05). In the correlation analysis, MMP9 levels in UUO groups had a negative correlation with the expression of TGF-ß1, VEGF, Col-IV, FN, and RIF index (all P<0.05). In UUO groups, VEGF levels had a positive correlation with the expression of TGF-ß1, Col-IV, FN, and RIF index (all P<0.05). In conclusion, with the aggravation of RIF lesions, MMP9 levels decreased, and VEGF levels increased. Whether there is a mutual inhibition relationship between them remains to be confirmed by further experiments.

Acupuncture promotes nerve repair through the benign regulation of mTOR-mediated neuronal autophagy in traumatic brain injury rats.

AIMS: Recent investigations have already proved the neuroprotective efficacy of acupuncture in clinical practice in the treatment of neurological diseases, such as traumatic brain injury (TBI). Since growing evidence has suggested that neuronal autophagy was involved in multiple stages of TBI, this study aims to clarify the autophagy mediating mechanism underlying the neuroprotective effect of acupuncture in TBI rats. METHODS: Three experiments were carried out to detect changes in neuronal autophagy and identify the potential molecular mechanism underlying the neuroprotective effect of acupuncture for TBI treatment. Feeney's free-falling epidural impingement method was used to establish the moderate TBI rat model; modified neurological severity scoring (mNSS) was used for neurological recovery evaluation. Nissl and HE staining were used to examine the histopathological changes. Immunofluorescence was used to detect the LC3-positive cell rate. The transmission electron microscope (TEM) was used to investigate the morphology and quantity of autophagosomes. Western blotting was used to determine the protein expressions of LC3, p62, beclin1, mTOR, ULK1, p-mTOR, and p-ULK1. Quantitative real-time polymerase chain reaction (qRT-PCR) was used for gene expressions analysis of LC3 mRNA and p62 mRNA. Co-immunoprecipitation (CO-IP) method was used to identify the protein interaction of mTOR and ULK1. RESULTS: On Day 3 after TBI, acupuncture accelerated the removal of damaged cellular structures by promoting neuronal autophagy; on Day 7 and Day 14 after TBI, acupuncture inhibited neuronal autophagy, preventing excessive autophagy and thus alleviated nerve damage. In addition, the simultaneous treatment with 3-MA or rapamycin at different stages after TBI attenuated the effect of acupuncture. CONCLUSION: Acupuncture has a benign regulatory effect on neuronal autophagy in different stages of TBI, possibly through the mTOR/ULK1 pathway.

Effects of acupuncture on microglial polarization and the TLR4/TRIF/MyD88 pathway in a rat model of traumatic brain injury.

OBJECTIVE: Neuroinflammation caused by traumatic brain injury (TBI) can lead to neurological deficits. Acupuncture can inhibit neuroinflammation and promote nerve repair; however, the specific mechanism is still unclear. The purpose of this study was to explore whether acupuncture could modulate the M1 and M2 phenotypic polarization of microglia in a rat model of TBI via the toll-like receptor 4 (TLR4)/intracellular toll-interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-ß (TRIF)/myeloid differentiation factor 88 (MyD88) pathway. METHODS: A total of 90 adult male Sprague-Dawley (SD) rats, SPF grade, were randomly divided into a normal group, model group and acupuncture group. Each group was further divided into three subgroups (first, third, and fifth day groups) according to the treatment time (n = 10 rats/subgroup). We used the modified neurological severity score (mNSS) method to quantify neurological deficits before and after modeling. We used Nissl staining to observe the pathological changes in brain tissue, flow cytometry to detect the proportion of M1 and M2 polarized microglia in the injured area on the first, third and fifth day, and co-immunoprecipitation (Co-IP) to examine TLR4/TRIF/MyD88 expression in microglia on the first, third and fifth day, as well as expression of the amount of binding of TLR4 with TRIF and MyD88. RESULTS: Compared to the model group, mNSS in the acupuncture group gradually decreased and pathological morphology improved. The proportion of CD11b/CD86 positive cells was decreased, while that of CD11b/CD206 was increased in the acupuncture group. Expression of IP TLR4, IP TRIF and IP MyD88 also decreased in the acupuncture group. CONCLUSION: The results of this study demonstrate that one of the mechanisms through which acupuncture mitigates neuroinflammation and promotes nerve repair in TBI rats may be inhibition of M1 phenotypic polarization and promotion of M2 phenotypic polarization through inhibition of the TLR4/TRIF/MyD88 signaling pathway.