Ursolic Acid Protects Neurons in Temporal Lobe Epilepsy and Cognitive Impairment by Repressing Inflammation and Oxidation.

Temporal lobe epilepsy (TLE) is characterized as an impaired ability of learning and memory with periodic and unpredictable seizures. Status epilepticus (SE) is one of the main causes of TLE. Neuroinflammation and oxidative stress are directly involved in epileptogenesis and neurodegeneration, promoting chronic epilepsy and cognitive deficit. Previous studies have shown that ursolic acid (UA) represses inflammation and oxidative stress, contributing to neuroprotection. Herein, we demonstrated that UA treatment alleviated seizure behavior and cognitive impairment induced by epilepsy. Moreover, UA treatment rescued hippocampal neuronal damage, aberrant neurogenesis, and ectopic migration, which are commonly accompanied by epilepsy occurrence. Our study also demonstrated that UA treatment remarkably suppressed the SE-induced neuroinflammation, evidenced by activated microglial cells and decreased inflammation factors, including TNF-α and IL-1ß. Likewise, the expression levels of oxidative stress damage markers and oxidative phosphorylation (OXPHOS) enzyme complexes of mitochondria were also remarkably downregulated following the UA treatment, suggesting that UA suppressed the damage caused by the high oxidative stress and the defect mitochondrial function induced by SE. Furthermore, UA treatment attenuated GABAergic interneuron loss. In summary, our study clarified the notable anti-seizure and neuroprotective properties of UA in pilocarpine-induced epileptic rats, which is mainly achieved by abilities of anti-inflammation and anti-oxidation. Our study indicates the potential advantage of UA application in ameliorating epileptic sequelae.

Higher rate of skin rash in a phase II trial with weekly nanoparticle albumin-bound paclitaxel and cisplatin combination in Chinese breast cancer patients.

BACKGROUND: The aim of this sub-study is to explore the incidence of skin rash among advanced breast cancer(ABC) patients in a phase II trial treated with weekly nab-paclitaxel and cisplatin combination. METHODS: Nab-paclitaxel(125 mg/m2) was administered on days 1, 8, 15, followed by cisplatin(75 mg/m2) on day 1 every 28 day cycle until disease progression, intolerable toxicities or the maximum of 6 cycles. Patients who received at least one injection of the study drug were included in this analysis of the incidence of skin rash among Chinese patients. Toxicity was graded using the CTCAE4.0 criteria. Statistical analysis was carried out by using SPSS 16.0 (SPSS Inc, Chicago, IL). RESULTS: Seventy three patients were enrolled and eligible for analysis. A total of 384 cycles were administered at the time of this analysis. Rash was presented in 27 patients (37.0%). The most common sites involved were face (14/27), neck (14/27), limbs (18/27) and frictional parts of the trunk (10/27). Macular and papular rash with pruritus commonly occurred 2 (95% CI: 1-7) days after the first day of chemotherapy. Only one patient developed Grade 3 skin toxicity with generalized erythroderma and disfigurement of the face requiring dose reduction. The rash gradually regressed 2 (95% CI: 1-10) days after antihistamines used, but pigmentation remained in 13/27 cases. The incidence rate of skin rash was significantly higher than what has been described for western patients (approximate 4%, P < 0.0001). CONCLUSION: A higher rate of maculo-papular rash occurred in Chinese breast cancer patients treated with weekly nab-paclitaxel compared to western patients. The albumin component of nab-paclitaxel might be the cause of the skin disorder. TRIAL REGISTRATION: NCT01149798.

Downregulation of survivin and activation of caspase-3 through the PI3K/Akt pathway in ursolic acid-induced HepG2 cell apoptosis.

Ursolic acid (UA), a naturally occurring pentacyclic triterpene, is a potent in-vitro anticancer agent, acting through control of growth, apoptosis and differentiation. As the mechanism of its proapoptotic effects on human hepatocellular carcinoma cells has not been extensively studied, we performed an in depth evaluation of the effects of UA on apoptosis in human HepG2 cells. UA was found to inhibit the proliferation of HepG2 cells in a concentration and time-dependent manner. After treatment, cells showed evidence of activation of apoptosis, including the presence of apoptotic bodies and DNA fragmentation. UA-induced apoptosis was accompanied by a significant decrease in bcl-2 and survivin expression, with the corresponding ratio of bax/bcl-2 increased. The treatment with UA also increased the protein level and enzymatic activity of caspase-3. Z-DEVD-fmk, a specific caspase-3 inhibitor, significantly inhibited both the cytotoxic effect and the DNA fragmentation induced by UA, demonstrating the requirement for caspase-3 activity in UA-induced apoptosis. Inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway was also involved, as inhibition of PI3K by LY294002 significantly increased UA-induced apoptosis. Kinetic experiments indicated that UA downregulated PI3K/p85 subunit (PI3K/p85) and phospho-Akt, before downregulating survivin. The further results also confirmed that LY294002 not only downregulated survivin alone, but considerably enhanced the repression of survivin combined with UA. UA therefore seemed to downregulate the expression of survivin by blocking PI3K/Akt. Taken together, the data suggest that the proapoptotic effect of UA on HepG2 cells is mediated by activation of caspase-3, and is highly correlated with inactivation of PI3K/Akt/survivin pathway.

Knockdown of survivin and upregulation of p53 gene expression by small interfering RNA induces apoptosis in human gastric carcinoma cell line SGC-823.

BACKGROUND: The survivin gene may be a good target for cancer gene therapy because it is overexpressed in a variety of human tumors, including gastric cancer, but not in differentiated adult tissues. To explore effects of the siRNA of the survivin gene inducing apoptosis in human gastric cancer cells, three siRNAs, cpusiRNA1, cpusiRNA2, and cpusiRNA3, were designed and transferred into human gastric carcinoma cell line SGC-823 (SGC-823). MATERIALS AND METHODS: The opposite livability on SGC-823 cells was assayed with an MTT test. The change of mRNA and protein of survivin and p53 gene were detected by reverse-transcriptase polymerase chain reaction and Western blot, respectively. Cell apoptosis was assayed by flow cytometry. RESULTS: The growth of SGC-823 cells decreased by 62.6%, 61.4%, and 55.1% when they were transfected with 400 nM of siRNA cpusiRNA1, cpusiRNA2, and cpusiRNA3 after 48 hours, in comparison to the control group. Also, the expression mRNA and protein of the survivin gene were knocked down, while the mRNA and protein of p53 gene were upregulated. SGC-823 cells presented an increase in apoptosis index. CONCLUSIONS: Small interfering RNA can exert a knockdown of the survivin gene expression and upregulation the p53 gene in SGC-823 cells and effectively induce apoptosis and inhibit the growth of human gastric carcinoma cell line SGC-823.

Knockdown of SMYD3 by RNA interference inhibits cervical carcinoma cell growth and invasion in vitro.

Elevated expression of SMYD3 is a frequent genetic abnormality in several malignancies. Few studies knocking down SMYD3 expression in cervical carcinoma cells have been performed to date. In this paper, we established an inducible short hairpin RNA expression system to examine its role in maintaining the malignant phenotype of HeLa cells. After being induced by doxycycline, SMYD3 mRNA and protein expression were both reduced, and significant reductions in cell proliferation, colony formation and migration/invasion activity were observed in the SMYD3-silenced HeLa cells. The percentage of cells in sub-G1 was elevated and DNA ladder formation could be detected, indicating potent induction of apoptosis by SMYD3 knockdown. These findings imply that SMYD3 plays crucial roles in HeLa cell proliferation and migration/invasion, and that it may be a useful therapeutic target in human cervical carcinomas.

Survivin gene RNA interference induces apoptosis in human HL60 leukemia cell lines.

BACKGROUND: It has been demonstrated that survivin, a member of the inhibitor of apoptosis (IAP) protein family, is expressed in human cancers but is undetectable in normal differentiated tissues. HL60 siRNA was introduced into HL60 cells to investigate its effect on cancer cell growth. METHODS: The opposite livability on HL60 cells was assayed with an MTT test. The change of mRNA and protein of the survivin gene were detected by reverse transcriptase polymerase chain reaction and Western blot, respectively. Cell apoptosis was assayed by flow cytometry. RESULTS: The growth of HL60 cells decreased by 65.3%, 62.1%, and 52.4% when they transfected with 400 nM siRNA lyh1, lyh2, and lyh3 after 48 hours, in comparison to the control group. Also, the mRNA and protein were knocked down and HL60 cells presented an increase in apoptosis index. CONCLUSIONS: Small interfering RNA can exert a knockdown of survivin gene expression in HL60 cells, and effectively induce apoptosis and inhibit the growth of leukemia cells.

[A universal high-throughput novel method of constructing the vectors].

In this paper, a simple universal high-throughput method of constructing the vectors was developed. It could add proper adapter when the PCR primers were designed, and the purpose fragments were cloned by PCR, and the various complementary sticky ends were created by T4 DNA polymerase's 3' -exodeoxyribonuclease activity. If all these fragments were put together with DNA ligase, they would recombinate in an orientation. If they had been transformated, the tansformants would be identified. Let's take the Oryza sativa single-cross homologous recombination chloroplast expression vector pRSMGA which was constructed with seven fragments as an example, if the vector pRSMGA was constructed in using the method what had mentioned, only twice recombination and transformation would be done. Scores of experiments had proved that it is a simple universal high-throughput novel method to construct the complicated vectors, which has not appeared in the periodical.

[Construction of tobacco chloroplast multicistron site integration expression vector and its transgene].

According to the published DNA sequence, a serial of elements for constructing the tobacco chloroplast multicistron site integrating expression vectors have been cloned by PCR technique, which include Prrn (a modified plastid ribosomal RNA operon promoter), psbA3' (the 3' region of the plastid psbA gene), aadA gene (encoding aminoglycoside 3'-adenylytransferase), man gene (encoding mannase), gfp gene (encoding green fluorescence protein) and tobacco chloroplast high-frequency homologous recombination ctDNA fragment (psaA/psbC, 3463 bp) (Fig.2). A tobacco chloroplast multicistron expression vector pLM4 (Fig.1) (-psaA-Prrn-SD-man-SD-gfp-SD-aadA-psbA3'- psbC-) was constructed with these elements. Then the tobacco leaves were bombarded 5 times with gold particles coated with the vector pLM4. After growing on the screening medium, the function of aadA gene was identified (Fig.3), and the function of gfp gene was confirmed by laser scanner (Fig.4), the expression of man was identified by Western blot (Fig.5). All these genes man, gfp and aadA being integrated in the tobacco chloroplast genome DNA were confirmed by PCR (Fig.6). And the multicistron expression cassette integrating in tobacco chloroplast genome DNA was confirmed by RFLP (Fig.7). All these showed that the three genes in the tomato vector pLM4 were expressed in tobacco chloroplast genome DNA.

[Study on the relationship between some genetic factors and peak bone mineral density in Beijing young women].

OBJECTIVE: To evaluate the relationship between the peak bone mineral density (PBMD) and vitamin D receptor (VDR), estrogen receptor (ER) allelic variants in Beijing young women. METHODS: From March, 2000 to July, 2001, one hundred and fifty-nine young healthy women (25 - 37 years old) in Beijing were voluntarily enrolled in the study. (1) BMD were measured by dual energy X-ray absorptiometry (DXEA) at lumbar and hip. (2) The polymorphism of VDR and ER genes were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). (3) The relationship between BMD and polymorphism of VDR and ER genes were examined. RESULTS: (1) Lumber BMD was positively correlated to height, weight and body mass index (BMI), whereas, the femoral neck BMD only to weight, and the other sites of hip BMD to BMI. (2) Although subjects with the VDR bb genotype had higher BMD than those with Bb genotype at lumber, femoral neck, inter and troch, no significant difference was found (P > 0.05). (3) In Ward triangle, subjects with ER PP genotype had significantly lower BMD than those in ER Pp and pp genotypes (P < 0.05). (4) Women with BbPP genotype combination had lower BMD levels at lumber and hip, and with bbPP and Bbpp genotypes combination significantly higher lumber BMD levels than BbPP genotype (P < 0.05). However, the differences of BMD among subjects with different VDR and ER genotypes became not significant after adjusting the confounder of body weight. CONCLUSIONS: (1) Body weight and BMI play important roles to PBMD of Beijing women. (2) There was no significant difference of BMD levels between VDR genotypes at any site. (3) PvuII polymorphism of ER gene was associated with low Ward triangle BMD. (4) There was significant relationship between the combination of ER and VDR polymorphisms at lumbar and hip BMD. Our data suggest that genetic variation at the ER locus, singly and in relation to the VDR locus, may influence the attainment and maintenance of peak bone mass in young women.