Puerarin Prevents LPS-Induced Osteoclast Formation and Bone Loss via Inhibition of Akt Activation.

Osteolysis induced by chronic Gram-negative bacterial infection underlies many bone diseases such as osteomyelitis, septic arthritis, and periodontitis. Drugs that inhibit lipopolysaccharide (LPS)-induced osteolysis are critically needed for the prevention of bone destruction in infective bone diseases. In this study, we assessed the effect of puerarin, a natural isoflavone isolated from Pueraria lobata OHWI root, on LPS-induced osteoclastogenesis and bone loss. Our in vitro study showed that puerarin significantly inhibited LPS-induced osteoclast differentiation from osteoclast precursor RAW264.7 cells. The inhibition occurred through suppressing the production of osteoclast activating factor tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and prostaglandin E2 (PGE2), which led to down-regulating mRNA expression of osteoclastogenic genes including tartrate-resistant acid phosphatase (TRAP), cathepsin K and matrix metalloprotein 9 (MMP-9). Furthermore, LPS triggered activation of Akt in osteoclast precursor RAW264.7 cells, which was inhibited by puerarin treatment. In vivo, puerarin attenuated LPS-induced bone loss in a murine calvarial osteolysis model. Collectively, puerarin prevents LPS-induced osteoclast formation, function and bone loss, where the inhibition of Akt activation plays an important role. These findings provide evidences that puerarin might be beneficial as a promising candidate drug for the prevention and treatment of bacteria-induced bone destruction disease, and give new insights for understanding its possible mechanism.

[Expression and Clinical Significance of N-cadherin in Bone Marrow Leukemic Cells Derived from Patients with Acute Leukemia].

OBJECTIVE: To investigate the expression of N-cadherin in bone marrow leukemic cells derived from acute leukemia patients and its clinical significances. METHODS: A total of 113 patients with acute leukemia were enrolled in this study. Flow cytometry was employed to detect the expression of N-Cadherin in bone marrow leukemic cells from acute leukemia patients and the relationships between the N-cadherin expression and the clinical characteristics of patients with acute leukemia were analyzed. RESULTS: The expression of N-Cadherin in bone marrow leukemic cells deriveted from patients with acute leukemia was variable with 0%-99.7%. For adult AML patients, the positive rate of CD34 in N-cadherin+ group was significantly higher than that in N-cadherin- group(67.39% vs 33.33%)(P=0.013), while the differences of total CR rate and rate of CR after 1 cycle of induction treatment were not significant between these 2 groups(P>0.05). As to ALL patients, N-cadherin+ group had significant lower WBC count (21.31±7.07 vs 51.10±23.69)(P=0.008) and lower percentage of peripheral blood blast (43.22±5.75% vs 66.45±5.65%)(P=0.015). The CR rate after 1 cycle of induction treatment and rate of overall CR were lower and the relapse rate was higher in N-cadherin+ ALL group than those in N-cadherin- ALL group, but the differences were not significant (P>0.05). For childhood ALL, the positive rate of CD33 in N-cadherin+ group was significantly higher than that in N-cadherin- group(47.62% vs 0%)(P=0.012). The relapse rate was higher in N-cadherin+ group than that in N-cadherin- group (30.00% vs 0%)(P=0.115). The median survival time, 3-year overall OS rate and 3-year relapse-free survival rate in N-cadherin- groups of adult AML, non-M3 AML, ALL and chidhood ALL paients were superior to N-cadherin+ groups, but the differences were not significant. CONCLUSION: The expression of N-cadherin in bone marrow leukemic cells relates to some clinical features of patients with acute leukemia and to some extent has inferior effect on survival of patients with acute leukemia.

[Expression of N-Cadherin in Patients with Multiple Myeloma and Its Clinical Significance].

OBJECTIVE: To investigate the expression of N-Cadherin in the patients with multiple myeloma (MM) and to explore its clinical significance. METHODS: A total of 64 patients with multiple myeloma were enrolled in this study. The expression of N-Cadherin in bone marrow CD38⁺/CD138⁺ cells from multiple myeloma patients was detected by flow cytometry. The relationship between N-Cadherin expression and clinical prognostic factors was analyzed. RESULTS: Among 64 cases of MM, the expression of N-Cadherin in 17 patients (26.56%) was high (> 20%), while that in 47 cases (73.44%) was low (< 20%); The differences of N-Cadherin expression in disease staging and classification, known prognostic factors, myeloma cell antigen expression and bone damage between patients with high and low N-Cadherin expression were not statistically different; the difference N-Cadherin expression in genetic abnormalities such as D13S319 deletion, RB1 deletion and IGH gene rearrangement between above-methioned two groups was not significant. The 1q21 amplification rate in the group with high expression of N-Cadherin was enhanced significently; the overall survival (OS) times of patients with abnormally high and low expression levels of N-Cadherin were 26.7 months and 55.5 months respectively, and the difference was statistically significant (P < 0.05). CONCLUSION: The high expression of N-Cadherin in multiple myeloma may be one of the indicator for poor prognosis of MM, which may be related with 1q21 amplification.

[Establishing a mouse model of Sertoli-cell-only syndrome by administration of busulfan].

OBJECTIVE: To establish a stable and reliable model of Sertoli-cell-only syndrome in mice. METHODS: We randomly divided 60 NIH mice into two groups of equal number to receive intraperitoneal injection of busulfan (30 mg/kg) and 30 or 60 minutes of testis cooling. At 2, 4 and 8 weeks after treatment, we recorded the survival rate of the mice, weight of the testis and Johnsen scores, and conducted quantitative analysis on the degrees of spermatogenetic failure. RESULTS: There were no significant differences in the baseline body weight and survival rate between the intervention and control groups (P > 0.05). At 4 and 8 weeks, the testis weight and Johnsen score were significantly lower in the intervention group than in the control ([0.04 +/- 0.01] g and [0.05 +/- 0.01] g vs [0.09 +/- 0.03] g and [0.11 +/- 0.02] g, P < 0.05; 3.86 +/- 0.50 and 2.70 +/- 0.67 vs 9.60 +/- 0.25 and 9.76 +/- 0.43, P < 0.01). At 2, 4 and 8 weeks, the testis weights were (0.07 +/- 0.02) g, (0.06 +/- 0.01) g and (0.09 +/- 0.01) g, respectively, in the 30-min cooling group and (0.05 +/- 0.01) g, (0.04 +/- 0.02) g and (0.04 +/- 0.02) g in the 60-min cooling group, significantly lower than in the control side at the same time points ([0.11 +/- 0.01] g, [0.11 +/- 0.01] g and [0.12 +/- 0.00] g) (P < 0.05), and the Johnsen scores were 4.70 +/- 0.67, 2.70 +/- 0.84 and 6.10 +/- 1.14 in the 30-min and 1.67 +/- 0.58, 1.20 +/- 0.45 and 1.00 +/- 0.00 in the 60-min cooling group, remarkably lower than in the control side (9.60 +/- 3.23, 9.60 +/- 0.55 and 9.70 +/- 0.45) (P < 0.01). Histopathological examination of the cooled testes revealed considerable atrophy of seminal tubules, necrosis of seminiferous epithelia and peritubular fibrosis. CONCLUSION: Administration of busulfan has no obvious influence on the survival of mice, and is a reliable method for constructing a mouse model of Sertoli-cell-only syndrome.

[DAZL gene polymorphisms and astheno-teratozoospermia].

OBJECTIVE: To investigate the association between single nucleotide polymorphism (SNP) of the DZAL gene in infertile Han Chinese males with astheno-teratozoospermia. METHODS: We collected semen samples from 173 infertile Han Chinese men with astheno-teratozoospermia (case group) and 175 age-matched normal male volunteers (control group) for semen routine and morphological analyses. We obtained genomic DNA, genotyped the polymorphisms of the DAZL gene A260G and A386G via the Sequenom MassARRAY system, and compared the frequencies of the genotypes between the case and control groups. RESULTS: The AA nucleotide variant was found in the A260G and A386G polymorphisms of the DZAL gene in both the cases and controls, but the heterozygous AG variant in neither. CONCLUSION: The A260G and A386G polymorphisms of the DAZL gene are not correlated with astheno-teratozoospermia-induced male infertility in the Han Chinese population, and therefore could not be considered as molecular markers of male infertility.

[Association of PRM1-190C- > A polymorphism with teratozoospermia].

OBJECTIVE: To investigate the association of single nucleotide polymorphism (SNP) of the Protamine 1 (PRM1) gene in infertile men with teratozoospermia. METHODS: We collected semen samples from 157 infertile men with teratozoospermia (case group) and 37 age-matched male volunteers (control group), and subjected them to morphological analysis. We extracted genome DNA, genotyped the polymorphism of the PRM1-190C- > A SNP (rs2301365) using the Sequenom MassARRAY system, compared the genotype frequencies between the case and control groups, and analyzed the sperm morphological parameters of different genotypes in the infertile males with teratozoospermia. RESULTS: The frequencies of the genotypes CC, CA and AA were 38.9% (61), 44.6% (70) and 16.6% (26) in the case group, as compared with 45.9% (17), 51.4% (19) and 2.7% (1) in the control, with that of AA significantly higher in the patients than in the volunteers (P<0.05). The frequencies of the alleles C and A were 57.6% and 42.4% in the former, with no significant differences from 71.6% and 28.4% in the latter (P>0.05). Nor were any statistically significant differences observed in sperm morphology parameters between the genotype CC and CA, AA and CA + AA in the male patients (P>0.05). CONCLUSION: The SNP of PRM1-190C- > A might be associated with teratozoospermia-induced male infertility in the Han Chinese. Although this SNP may attribute to abnormal sperm morphology, the targeted part of sperm remains unclear.

[Differentially expressed genes in asthenospermia: a bioinformatics-based study].

OBJECTIVE: To study the differentially expressed genes in asthenospermia to gain a deeper insight into the molecular mechanisms of the disease. METHODS: We analyzed the differentially expressed genes in asthenospermia using GATHER, PANTHER and ToppGene online bioinformatics tools. RESULTS: Our bioinformatics mining and analyses revealed that the differentially expressed genes in asthenospermia played important roles in the cellular protein and macromolecular metabolism, protein modification, cell death, cell apoptosis and apoptosis induction. CONCLUSION: Asthenospermia patients experience a decline in sperm activity and the basic life activities of sperm simultaneously, and are also prone to cell apoptosis or death. Such differentially expressed genes as KIF3B, MYO15A, KIF6, KIF26B, KIF3A, DNHD2, DMN, DYNC2H1, STARD9, MYOHD1, and TPM1, which are involved in cytoskeletal structure, microtubule movement and cell movement, may be associated with asthenospermia, and therefore deserve further studies.

[Expressions of cysteine-rich secretory protein 2 in asthenospermia].

OBJECTIVE: To investigate the mRNA and protein expression levels of cysteine-rich secretory protein 2 (CRISP2) in the sperm of asthenospermia patients, and explore their relationship with sperm motility and related molecular mechanism. METHODS: We collected 78 semen samples from adult male patients with asthenospermia and another 70 from healthy volunteers as controls. We extracted total RNA and total protein from the sperm following purification of the sperm by Percoll gradient centrifugation, and detected the relative expressions of CRISP2 mRNA and protein in the two groups by RT-PCR, SYBR Green real-time PCR and Western blot. RESULTS: The expression of CRISP2 mRNA was down-regulated by 4.3 times and that of the CRISP2 protein by 1.71 times in the asthenospermia patients, significantly lower than in the normal control group (P < 0.05). CONCLUSION: The down-regulation of CRISP2 mRNA and protein expressions in the sperm of asthenospermia patients may be closely related with decreased sperm motility, which suggests that CRISP2 may serve as a potential molecular target for the research of asthenospermia.