Mesaconine alleviates doxorubicin-triggered cardiotoxicity and heart failure by activating PINK1-dependent cardiac mitophagy.

Aberrant mitophagy has been identified as a driver for energy metabolism disorder in most cardiac pathological processes. However, finding effective targeted agents and uncovering their precise modulatory mechanisms remain unconquered. Fuzi, the lateral roots of Aconitum carmichaelii, shows unique efficacy in reviving Yang for resuscitation, which has been widely used in clinics. As a main cardiotonic component of Fuzi, mesaconine has been proven effective in various cardiomyopathy models. Here, we aimed to define a previously unrevealed cardioprotective mechanism of mesaconine-mediated restoration of obstructive mitophagy. The functional implications of mesaconine were evaluated in doxorubicin (DOX)-induced heart failure models. DOX-treated mice showed characteristic cardiac dysfunction, ectopic myocardial energy disorder, and impaired mitophagy in cardiomyocytes, which could be remarkably reversed by mesaconine. The cardioprotective effect of mesaconine was primarily attributed to its ability to promote the restoration of mitophagy in cardiomyocytes, as evidenced by elevated expression of PINK1, a key mediator of mitophagy induction. Silencing PINK1 or deactivating mitophagy could completely abolish the protective effects of mesaconine. Together, our findings suggest that the cardioprotective effects of mesaconine appear to be dependent on the activation of PINK1-induced mitophagy and that mesaconine may constitute a promising therapeutic agent for the treatment of heart failure.

Mechanism of subchronic vinyl chloride exposure combined with a high-fat diet on hepatic steatosis.

Vinyl chloride (VC) is a common industrial organic chlorine and environmental pollutant. In recent years, the dietary structure of residents especially Chinese has gradually shifted to western dietary patterns. VC aggravates dietary fatty acid-induced hepatic steatosis, but its mechanism is still unclear. And if the risk factors for steatosis persist, more severe diseases such as fibrosis and cirrhosis will occur. Therefore, we studied the effects and mechanisms of VC (160 and 800 mg/m3 ) and its metabolite (chloroacetaldehyde, 2.25, 4.5, and 9 µM) on hepatic steatosis of high-fat diet (HFD)-fed mice and palmitic acid (PA, 100 µM) treated HepG2 cells. Liver and serum biochemical indicators and pathological staining of the liver showed that the hepatic steatosis of VC combined with HFD groups was more severe than that of single-exposure groups (HFD group, low-dose VC group, and high-dose VC group). Moreover, VC enhanced HFD-induced oxidative stress (OS) and endoplasmic reticulum stress (ERS) and further upregulated the expression of sterol regulatory element-binding protein 1 (SREBP-1) and FAS. Besides, antioxidants and ERS inhibitors reduced the steatosis of HepG2 cells induced by VC metabolites and PA. These results suggest that VC exposure can enhance the degree of hepatic steatosis in HFD-fed mice. VC combined with HFD led to OS and ERS and upregulated the expression of de novo lipogenesis-related proteins, which may be related to the occurrence of hepatic steatosis. And the increased expression of CYP2E1 induced by VC combined with HFD may be the cause of OS.

Mechanism investigation of ethosomes transdermal permeation.

Ethosomes are widely used to promote transdermal permeation of both lipophilic and hydrophilic drugs, but the mechanism of interaction between the ethosomes and the skin remains unclear. In this work, it was exploded with several technologies and facilities. Firstly, physical techniques such as attenuated total reflectance fourier-transform infrared and laser confocal Raman were used and the results indicated that the phospholipids configuration of stratum corneum changes from steady state to unstable state with the treatment of ethosomes. Differential scanning calorimetry reflected the thermodynamics change in stratum corneum after treatment with ethosomes. The results revealed that the skin of Bama mini-pigs, which is similar to human skin, treated by ethosomes had a relatively low Tm and enthalpy. Scanning electron microscopy and transmission electron microscopy showed that the microstructure and ultrastructure of stratum corneum was not damaged by ethosomes treatment. Furthermore, confocal laser scanning microscopy revealed that lipid labeled ethosomes could penetrate the skin via stratum corneum mainly through intercellular route, while during the process of penetration, phospholipids were retained in the upper epidermis. Cell experiments confirmed that ethosomes were distributed mainly on the cell membrane. Further study showed that only the drug-loaded ethosomes increased the amount of permeated drug. The current study, for the first time, elucidated the mechanistic behavior of ethosomes in transdermal application from molecular configuration, thermodynamic properties, ultrastructure, fluorescent labeling and cellular study. It is anticipated that the approaches and results described in the present study will benefit for better design of drug-loaded ethosomes.

Evidence based anti-osteoporosis effects of Periplaneta americana L on osteoblasts, osteoclasts, vascular endothelial cells and bone marrow derived mesenchymal stem cells.

BACKGROUND: Kangfuxin (KFX) is the ethanol extract of Periplaneta americana L, which has been widely used in the Traditional Chinese Medicine for the repair and regeneration of injured organ and tissues with long history. This study is to investigate the influence of KFX in the various cellular activities and evaluate the anti-osteoporosis potential of KFX. METHODS: The influence of the KFX in the cellular activities, including: 1) migration, osteocalcin secretion of osteoblasts; 2) apoptosis of osteoclasts; 3) migration and tube formation of human umbilical vein endothelial cell (HUVEC); and 4) proliferation, cell cycle regulation and migration of bone marrow mesenchymal stem cells (BMSCs), were investigated systematically. RESULTS: KFX was shown to significantly 1) Promote of the migration of osteoblasts, HUVEC, and BMSCs; 2) Increase the secretion of osteocalcin and mineralization of osteoblasts; 3) Accelerate the apoptosis of osteoclasts; 4) Stimulate the proliferation and regulate the cell cycle of BMSCs. CONCLUSION: Taken together, these results provide the evidence for the osteogenesis, anti-osteoporosis and angiogenesis effects of KFX, with the mechanism of activating the bone formation through stimulating the osteoblasts and HUVECs, as well as inhibiting the bone absorption by inhibiting the osteoclasts activities. The KFX was definitely shown a promising bone turnover agent with great potential for anti-osteoporosis treatment.

Codelivery of curcumin and doxorubicin by MPEG-PCL results in improved efficacy of systemically administered chemotherapy in mice with lung cancer.

Systemic administration of chemotherapy for cancer often has toxic side effects, limiting the doses that can be used in its treatment. In this study, we developed methoxy poly(ethylene glycol)-poly(caprolactone) (MPEG-PCL) micelles loaded with curcumin and doxorubicin (Cur-Dox/MPEG-PCL) that were tolerated by recipient mice and had enhanced antitumor effects and fewer side effects. It was shown that these Cur-Dox/MPEG-PCL micelles could release curcumin and doxorubicin slowly in vitro. The long circulation time of MPEG-PCL micelles and the slow rate of release of curcumin and doxorubicin in vivo may help to maintain plasma concentrations of active drug. We also demonstrated that Cur-Dox/MPEG-PCL had improved antitumor effects both in vivo and in vitro. The mechanism by which Cur-Dox/MPEG-PCL micelles inhibit lung cancer might involve increased apoptosis of tumor cells and inhibition of tumor angiogenesis. We found advantages using Cur-Dox/MPEG-PCL micelles in the treatment of cancer, with Cur-Dox/MPEG-PCL achieving better inhibition of LL/2 lung cancer growth in vivo and in vitro. Our study indicates that Cur-Dox/MPEG-PCL micelles may be an effective treatment strategy for cancer in the future.

[Effect of BYDV-MP nuclear localization signal on the movement of PVX].

Abstract:By using PVX derived vector pGR107, the effect of BYDV-MP nuclear localization signal on the movement of PVX was studied. BYDV-MP was cloned into pGR107 using GFP as an indicator. BYDV-MP was then shown to induce the systemic infection and exacerbate the symptom of PVX through infecting Nicotiana benthamiana. When the PVX gene encoding 25kD protein, which functioned as a systematic movemnet protein,was deleted and the above experiment was repeated, the result showed that BYDV-MP could compensate the systemic movement of PVX. A serial mutants with substitutions on the fifth, sixth and seventh amino acids of BYDV-MP nuclear localization signal was further constructed. It was found that the mutants at the fifth, sixth amino acids in BYDV-MP nuclear localization signal could only delay or weaken systemic movement of PVX whereas the mutant at seventh amino acid could entirely inhibit systemic movement of PVX.

Adenovirus-mediated and tumor-specific transgene expression of the sodium-iodide symporter from the human telomerase reverse transcriptase promoter enhances killing of lung cancer cell line in vitro.

BACKGROUND: The sodium-iodide symporter (NIS) protein can mediate the active radioiodine uptake. The human telomerase reverse transcriptase (hTERT) promoter is known to be selectively reactivated in majority of tumors and hence could be used for tumor targeting. We constructed a recombinant adenovirus containing the human sodium iodide symporter (hNIS) gene directed by the hTERT promoter, characterized the ability of infected cells in uptaking iodide, and explored the therapeutic efficacy of (131)I in a lung cancer cell line in vitro. METHODS: The hTERT promoter was amplified by PCR from DNA isolated from log-phase HepG2 cells, subcloned into lineralized FL*-hNIS/pcDNA3, and then the hTERT-hNIS sequence was subcloned into the shuttle plasmid pAdTrack. The recombinant adenovirus Ad-hTERT-hNIS was constructed by AdEasy system. A positive control adenovirus Ad-CMV-hNIS and a negative control adenovirus Ad-CMV were created similarly. A549 cells were transduced with recombinant adenoviruses. (125)I uptake studies and sodium perchlorate suppression studies were used to confirm hNIS expression and function. Toxic effects of (131)I on tumor cells were studied by in vitro clonogenic assay. RESULTS: We first successfully constructed an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter. When infected with recombinant adenovirus constructs expressing hNIS directed by hTERT- and CMV-promoters (Ad-hTERT-hNIS and Ad-CMV-hNIS, respectively), the lung cancer cell line A549 had increased ability to uptake radioiodide up to 23- and 30-fold compared to the control parental cells, respectively. The radioiodide uptake ability of both the Ad-CMV-hNIS and Ad-hTERT-hNIS transduced cell lines were repressed 11-fold by sodium perchlorate (NaClO4). The subsequent in vitro clonogenic assay of the infected A549 cell line was further repressed to 23% (Ad-CMV-hNIS) and 30% (Ad-hTERT-hNIS) of the control group after receiving radioiodide for 7 hours (P < 0.001). CONCLUSION: Our preliminary study indicates that an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter has the potential to become an effective wide-spectrum yet highly specific anti-cancer strategy.

Growth inhibition induced by short hairpin RNA to silence survivin gene in human pancreatic cancer cells.

BACKGROUND: Survivin is known to be overexpressed in various human malignancies, including pancreatic cancer, and mediates cancer cell proliferation and tumor growth, so the regulation of this molecule could be a new strategy for treating pancreatic cancer. In this study, short hairpin RNAs (shRNAs) specific to survivin were introduced into human pancreatic cancer Patu8988 cells to investigate the inhibitory effects on survivin expression and cell proliferation in vitro and in vivo. METHODS: Three kinds of shRNA specific to the survivin gene were designed and cloned into eukaryotic expression plasmid pGenesil-1 vector. Subsequently the recombinant plasmids were transfected into human pancreatic cancer Patu8988 cells with lipfectamineTM 2000 reagent. The mRNA and protein expressions of survivin in the transiently transfected Patu8988 cells were determined by RT-PCR, flow cytometry, and Western blotting analysis. The proliferation inhibition rates of stably transfected Patu8988 cells were determined by MTT assay. The antitumor activities of the three kinds of survivin-shRNA plasmids were evaluated in BALB/c nude mice inoculated with Patu8988 cells and bearing human pancreatic cancer. RESULTS: The three survivin-shRNA plasmids named pGenesil-1-survivin-1, pGenesil-1-survivin-2 and pGenesil-1-survivin-1+2 (with double interfering RNA sites) were successfully constructed, and were confirmed by restriction enzyme cutting and sequencing. At 48 hours after transfection, the expression of survivin mRNA and protein was inhibited in Patu8988 cells transfected with pGenesil-1-survivin-1, pGenesil-1-survivin-2, and pGenesil-1-survivin-1+2 when compared with that of either pGenesil-1-NC (with scrambled small interfering RNA) transfected cells or control cells (P<0.05). The MTT results showed that the proliferation rates of Patu8988 cells stably transfected with survivin-shRNA plasmids were reduced when compared with that of either pGenesil-1-NC transfected cells or control cells (P<0.01). Furthermore, when Patu8988 cells stably transfected with survivin-shRNA were injected into BALB/c nude mice, tumor growth was dramatically lower and the tumor was smaller than that of either pGenesil-1-NC transfected cells or control cells (P<0.01). The inhibitory effect of pGenesil-1-survivin-1 was the best among the three kinds of survivin-shRNA plasmids, but no combination of inhibitory effects was found in pGenesil-1-survivin-1+2. CONCLUSIONS: shRNAs specific to survivin have gene silencing effects and inhibit pancreatic cancer cell proliferation. shRNA activity against survivin could be of potential value in gene therapy for pancreatic cancer. However, shRNAs with double combining sites did not significantly enhance the interference compared with single site shRNAs, therefore further studies on this are needed.

[Generation, screening and preliminary identification of specific Fab phage antibody library against Daudi cell strain].

AIM: To generate and screen the specific Fab phage antibody library against human Daudi cell strain in B-lymphoma and identify the positive clones. METHODS: BALB/c mice were immunized with Daudi cells, and antisera were titrated by ELISA. Following the demonstration of sufficient antibody titer, total RNA was extracted from splenic lymphocytes of the immunized mice and RT-PCR was used to amplify kappa light chain and Fd fragments of heavy chain. After restrictive digestion with Sac I/Xba I and Xho I/Spe I, the kappa light chain and the Fd fragments were successively inserted into the phagemid vector pComb3H-SS and then electroporated into E.coli XL1-Blue. The specific Fab phage antibody library against Daudi cell strain in human B-lymphoma was constructed by infection of helper phage VCSM13. Following six rounds of biopanning with Daudi cells, the antigen binding activities of random clones were tested by ELISA to select the positive clones, which were further DNA sequenced, expressed in E.coli XL1-Blue and identified by Western blot. RESULTS: The Fab phage antibody library with 3.13x10(7) size was constructed and four positive clones which specifically recognized Daudi cell strain were isolated. In amino acid sequences, the variable heavy domains (V(H)) were found to be 80%-02.394% and variable light domains (V(L)) 88%-95% homologous with respective murine germline genes in GenBank. Furthermore, soluble Fab antibodies of the positive clones were successfully expressed in E.coli XL1-Blue and the reactivity with the membrane proteins of Daudi cells was demonstrated by Western blot. CONCLUSION: Fab phage antibody library is successfully constructed and specific antibodies against membrane antigens in Daudi cells are obtained, which provides an experimental foundation for the further investigation of B-lymphoma immunotherapy.

Enhanced therapeutic effects for human pancreatic cancer by application K-ras and IGF-IR antisense oligodeoxynucleotides.

AIM: To investigate the combined effects of K-ras antisense oligodeoxynucleotide (K-ras ASODN) specific to GTT point mutation at codon 12 and type I insulin-like growth factor receptor (IGF-IR) antisense oligodeoxynucleotide (IGF-IR ASODN) on proliferation and apoptosis of human pancreatic cancer Patu8988 cells in vitro and in vivo. METHODS: K-ras gene point mutation and its style at codon 12 of human pancreatic cancer cell line Patu8988 were detected by using polymerase chain reaction with special sequence primers (PCR-SSP) and sequence analysis. According to the mutation style, K-ras mutation ASODN specific to K-ras point mutation at codon 12 was designed and composed. After K-ras ASODN and IGF-IR ASODN treated on Patu8988 cells respectively or cooperatively, the proliferation and morphological change of Patu8988 cells were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony forming assay and transmission electron microscopy; the expression of K-ras and IGF-IR mRNA and protein in the treated cells was measured by reverse-transcript polymerase chain reaction (RT-PCR) and flow cytometry respectively; apoptosis was determined by flow cytometry. The combined antitumor activity of K-ras ASODN and IGF-IR ASODN was evaluated in BALB/c nude mice bearing human pancreatic cancer inoculated with Patu8988 cells. RESULTS: The results of PCR-SSP and sequence analysis showed that the human pancreatic cancer cell line Patu8988 had point mutation at codon 12, and the mutation style was GGT-->GTT. 2-32 microg/mL K-ras ASODN and 2-32 microg/mL IGF-IR ASODN could inhibit Patu8988 cells' growth, induce apoptosis and decrease the expression of K-ras and IGF-IR mRNA and protein alone. However, there was much more effective inhibition of growth and induction of apoptosis by their combination than by each one alone. In tumor bearing mice, the combination of K-ras ASODN and IGF-IR ASODN showed a significant inhibitory effect on the growth of transplanted pancreatic cancer, resulting in a statistically significant difference compared with each alone. CONCLUSION: It has been found that K-ras ASODN combined with IGF-IR ASODN could cooperatively inhibit the growth of Patu8988 cells, and induce their apoptosis via reinforcing specific down regulation of K-ras and IGF-IR mRNA and protein expression.

[Combinational effects of K-ras and IGF-IR antisense oligodeoxynucleotide on proliferation and apoptosis of human pancreatic cancer Patu8988 cells].

BACKGROUND & OBJECTIVE: Point mutation of K-ras gene and overexpression of insulin-like growth factor receptor type 1 (IGF-IR) may contribute to the progression and aggressiveness of pancreatic cancer. Antisense oligodeoxynucleotide (ASODN) against K-ras mRNA and IGF-IR mRNA may inhibit the proliferation of pancreatic cancer cells. This study was to investigate the combinational effects of K-ras ASODN and IGF-IR ASODN on proliferation and apoptosis of human pancreatic cancer Patu8988 cells in vitro and in vivo. METHODS: K-ras gene point mutation in Patu8988 cells was detected by polymerase chain reaction using special sequence primers (PCR-SSP) and sequence analysis. According to the mutation style, K-ras ASODN was designed and composed. K-ras ASODN and IGF-IR ASODN were transfected into Patu8988 cells alone or in combination. Cell proliferation was analyzed by MTT and colony forming assay. The morphologic changes of Patu8988 cells were assessed under transmission electron microscope. The expression of K-ras and IGF-IR mRNA and protein in Patu8988 cells was measured by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM). Cell apoptosis was determined by FCM. The combinational antitumor activity of K-ras ASODN and IGF-IR ASODN was evaluated in BALB/c nude mice bearing human pancreatic cancer inoculated with Patu8988 cells. RESULTS: The point mutation of K-ras gene at codon 12 was detected in Patu8988 cells, and the mutation style was GGT-->GTT. Either 2-32 microg/mL K-ras ASODN or IGF-IR ASODN inhibited proliferation and induced apoptosis of Patu8988 cells. This effect was more obvious when K-ras ASODN and IGF-IR ASODN were used in combination than used alone (P<0.01). In tumor-bearing mice, the inhibitory effect on the growth of transplanted pancreatic cancer was more obvious when K-ras ASODN and IGF-IR ASODN were used in combination than used alone (P<0.01). CONCLUSION: K-ras ASODN combined with IGF-IR ASODN could cooperatively inhibit the proliferation of Patu8988 cells and induce their apoptosis via down-regulating K-ras and IGF-IR expression.

[Combinational effects of bcl-2 antisense oligodeoxynucleotide and Rituximab on proliferation and apoptosis of B-lymphoma Raji cells].

AIM: To study the combinational effects of bcl-2 antisense oligodeoxynucleotide (bcl-2 ASODN) and Rituximab (anti-CD20 monoclonal antibody) on proliferation and apoptosis of B-lymphoma Raji cells in vitro and in vivo, and to explore the possible mechanisms. METHODS: After bcl-2 ASODN and Rituximab treating on Raji cells respectively or cooperatively, the proliferation of Raji cells, the expression of bcl-2 protein, the apoptosis and the expression of bcl-2 mRNA were detected by MTT assay, flow cytometry (FCM) and RT-PCR, respectively. The combinational antitumor activity of bcl-2 ASODN and Rituximab was evaluated in BALB/c nude mice bearing B-lymphoma inoculated with Raji cells. RESULTS: 5-30 micromol/L bcl-2 ASODN and 1-16 mg/L Rituximab could inhibit Raji cells' growth, induce apoptosis and decrease the expression of bcl-2 protein and bcl-2 mRNA alone. But there was much more effective inhibition of growth and induction of apoptosis by their combination than alone (P<0.01). In tumor bearing mice, combination of bcl-2 ASODN and Rituximab showed a significant inhibitory effect on the growth of transplanted B-lymphoma, resulting in a statistically significant difference compared with alone (P<0.01). CONCLUSION: It has been found that bcl-2 ASODN combined with Rituximab could significantly inhibit the growth of Raji cells and induce their apoptosis via reinforcing specific down regulation of bcl-2 protein and bcl-2 mRNA expression.

[Construction and screening of the specific Fab phage antibody library against Raji cell strain].

AIM: To construct and screen the specific Fab phage antibody library against human Raji cell strain in B-lymphoma. METHODS: BALB/c mice were immunized with Raji cells, and the antibody light chain kappa genes and heavy chain genes Fd from the spleen cells were amplified by RT-PCR. After restrictive digestion with Sac I/Xba I and Xho I/Spe I, the light chain kappa genes and heavy chain genes Fd were inserted into the phagemid vector pComb3H-SS successively and then electroporated into E.coli XL1-Blue. The specific Fab phage antibody library against Raji cell strain in human B-lymphoma was constructed by infection of helper phage VCSM13. The specific antibodies against Raji cells were obtained after selected with Raji cells. The binding activity with antigens was identified by ELISA and the positive clones were sequenced. RESULTS: The Fab phage antibody library with 2.18 x 10(7) volume was constructed and eight positive clones which specifically recognized Raji cell strain were isolated. Sequence analysis of the two positive clones showed that the variable heavy domains (VH) and variable light domains (VL) were highly homologous with the registered murine Ig heavy chain V region sequences and kappa light chain sequences, respectively. CONCLUSION: Fab phage antibody library was successfully constructed and specific antibodies against membrane antigens in Raji cells were obtained, which will provide an experimental foundation for the further investigation of B-lymphoma immunotherapy.

[Detection of 20q(-) chromosome abnormality in myelodysplastic syndrome by interphase fluorescence in situ hybridization].

In order to explore the value of interphase fluorescence in situ hybridization (FISH) in the detection of partial deletion of the long arm of chromosome 20 (20q(-)) in patients with myelodysplastic syndrome (MDS), spectrum Green fluorescein directly labeled yeast artificial chromosome (YAC) clone 912C3 which spans the breakpoint cluster region in band 20q12 was used as probes to perform interphase FISH on the marrow cells from 52 cases of MDS and 5 normal controls. 200 to 300 cells were scored for each case and cases which had cells with a green hybridization signal>7.16% were defined as 20q(-) positive. The results of FISH were compared with those of conventional cytogenetics (CC) assay. The results showed that among 52 cases of MDS, 7 (13.5%) cases were positive by FISH, however, of which, 4 cases were positive and the other 3 cases were negative by CC assay. It is concluded that YAC912C3 and interphase FISH providing a powerful technique in the detection of 20q(-) in MDS is an important complement to CC assay.