Puerarin promotes the viability and differentiation of MC3T3-E1 cells by enhancing LC3B-mediated autophagy through downregulation of miR-204.

Puerarin is a bioactive substance extracted from Pueraria lobata. It is known to promote the viability, differentiation and mineralization of osteoblasts. However, the molecular mechanisms involved in these activities are not well understood. The present study was conducted with the aim of elucidating the effect of puerarin on osteoblasts and to explore the underlying mechanism. CCK-8 analysis showed that puerarin (0.1, 1 and 10 µM) promoted the viability of osteoblastic MC3T3-E1 cells, with 1 µM of puerarin exhibiting the strongest effect. Moreover, 1 µM puerarin significantly increased the activity of alkaline phosphatase (ALP) and the formation of mineralized nodules in the MC3T3-E1 cells. Treatment with 1 µM puerarin for 72 h led to a significant upregulation in the expression level of microtubule-associated light chain 3 (LC3)B and Beclin1 proteins. This treatment was more effective in promoting LC3B expression than what was observed following treatment with rapamycin (overexpression for autophagy). The bilayer membrane structure of autophagosomes was observed by electron microscopy. Conversely, 3-methyladenine (3-MA, inhibitor of autophagy) reduced the cell viability as well as the activity of alkaline phosphatase (ALP) in MC3T3-E1 cells, although, there was no significant influence on mineralization. Prediction results of the biological information showed that LC3B could be a direct target of microRNA-204 (miR-204). In the present study, the expression level of miR-204 was decreased by puerarin. miR-204 mimics significantly decreased LC3B expression and inhibited auotophagosome formation, while the miR-204 inhibitor had the opposite effects. To conclude, the results of the present study suggest that puerarin promotes the viability and differentiation of MC3T3-E1 cells through autophagy, which is possibly associated with miR-204-regulated LC3B upregulation.

Puerarin promotes the viability and differentiation of MC3T3­E1 cells by miR­204­regulated Runx2 upregulation.

Puerarin has attracted increasing attention because of its beneficial effects on anti­osteoporosis, but the molecular mechanisms underlying its actions on osteoblasts are not fully understood. The current study aimed to investigate the effect of puerarin on the cell viability and differentiation of mouse MC3T3­E1 osteoblast­like cells in vitro and its underlying mechanisms. The results indicated that 0.01, 0.1 and 1 mg/ml puerarin significantly promoted the viability of osteoblasts, enhanced alkaline phosphatase (ALP) activity and increased the expression of transforming growth factor­ß1, Smad2, Smad3 and Runt­related transcription factor (Runx)2. Micro (mi)RNA target prediction programs predicted that miR­204 may directly target Runx2. Following treatment with 0.1 mg/ml puerarin for 48 h, the expression level of miR­204 was downregulated. Besides, miR­204 dramatically repressed the luciferase activity of wildtype Runx2 3'­UTR transfected cells, but not that of the mutant ones. Overexpression of miR­204 in osteoblasts significantly decreased the protein expression of Runx2, while inhibition of miR­204 enhanced Runx2's expression. In addition, overexpression of miR­204 inhibited the cell viability and ALP activity of osteoblasts, while inhibition of miR­204 had the opposite effect. The results suggested that puerarin may promote MC3T3­E1 osteoblast­like cell viability and differentiation, which may be related to the downregulation of miR­204 and the following activation of Runx2.

[Effects of fermented cattle dung on the growth and development of Tenebrio molitor larvae].

In order to make use of and industrialize the animal dung from large cattle farms, this paper explored the feasibility of using Tenebrio molitor to digest and utilize cattle dung. Cattle dung was mixed with the conventional feed (65% wheat bran, 30% corn flour, and 5% bean pulp) of T. molitor in definite proportions, and fermented with effective microorganisms (EM). The fermented products containing 60% and 80% of cattle dung (FD1 and FD2, respectively) were selected to feed T. molitor larvae, and the effects of the fermented products on the growth curve, death rate, pupation rate, and antioxidant system of the larvae were compared. Compared with CK (conventional deed), the FD1 made the developmental duration of the larvae prolonged by 10 days and the larvae's death rate upraised somewhat, but made the single larva's total food intake, average body mass, crude fat content, and ratio of unsaturated to saturated fat acids increased by 49%, 28%, 26%, and 32%, respectively (P < 0.05), and the activity of larvae's antioxidant system improved significantly, showing a remarkable adaptability of the larvae to FD1. Unlike FD1, FD2 displayed definite disadvantages in most test growth indicators, as compared with CK, indicating that T. molitor larvae had weak adaptability to FD2. Our findings suggested that using FD1 to feed the 3rd instar of T. molitor larvae would have good practical prospects in industrializing cattle dung.

[Density fluctuation of microfilariae and the role of residual infection source in filariasis transmission after its interruption].

OBJECTIVE: To investigate the density fluctuation of microfilariae, persistence of microfilaremia and possible new infection due to residual microfilaremia in areas with filariasis transmission interrupted. METHODS: The observation site was made in a village of Jishou City, Hunan Province. Inhabitants were regularly examined by thick blood smear and the density fluctuation of residual microfilaremia in known and newly-found cases were followed up. With a consent from the cases with residual microfilaremia, no treatment was given until they naturally turned negative. Antifilarial antibody level was detected by IFAT and a test kit for filariasis-special IgG4. Culex quinquefociatus was dissected to determine the natural infection rate and density of III stage filarial larvae in transmission season. The identified cases were followed-up by interviews and physical examinations to see if clinical manifestations appeared. RESULTS: Blood examination was carried out for all inhabitants for 10 times, 4 cases with microfilaremia, including 3 cases found at the beginning of the project and one newly infected case, were discovered after the interruption of filariasis transmission in the 19-year period. Among the 4 cases followed up, one case naturally turned negative within 7 years, one case became negative in the 9th year but returned positive in the 12th year, and then naturally turned negative in the 13th year. The 3rd case turned negative in the 14th year and was again positive in the 19th and the 20th years, and became negative through diethylcarbamazine (DEC) treatment in the 21st year. The new case was found to have microfilaremia in the 16th year and kept positive for 5 years until DEC treatment. Serological tests (IFAT and special IgG4) revealed no new positive cases. The natural infection rate and larvae density in Culex quinquefasciatus decreased annually. Conclusion The persistence period of residual microfilaremia in individual cases might last for more than 20 years after filariasis transmission has been interrupted.

[Prokaryotic expression and detective application of the main antigenic region of VP2 protein of Aleutian mink disease parvovirus].

To research safer diagnosis antigen for ADV, the main antigenic region VP2a and VP2b gene of ADV were obtained by restriction digestion of the recombinant plasmids pMD-VP2a and pMD-VP2b. Then the genes were respectively cloned into pMAL-c2 to get two prokaryotic recombinant plasmids pMAL-VPa and pMAL-VPb. The target genes were successfully expressed in the host cell TB1 when induced by IPTG. The Western blot analysis proved the recombinant proteins have good antigenic. The recombinant proteins were purified by KCL dyeing method, and were used as antigen to establish VP2-CIEP for AD diagnoses. The detection result shared 94.3% identity with that of CIEP. The results reported here show that VP2-CIEP is highly sensitive and specific and can benefit the research on the serodiagnosis to AD.

Primary survey of avian influenza virus and Newcastle disease virus infection in wild birds in some areas of Heilongjiang Province, China.

Two hundred thirty specimens of wild birds were collected from some areas in Heilongjiang Province during the period of 2003-2004, including two batches of specimens collected randomly from a same flock of mallards in Zhalong Natural Reserve in August and December, 2004, respectively. Primary virus isolation and identification for avian influenza virus (AIV) and Newcastle disease virus (NDV) were performed. The results showed that only two specimens of young mallards collected from Zhalong Natural Reserve in August, 2004 were positive to AIV (isolation rate 0.9%), and one strain (D57) of these two virus isolates was identified to be H9 subtype by hemagglutination inhibition test. Meanwhile, the two batches of blood serum samples of mallards from Zhalong were also examined for antibodies against AIV and NDV. Among 38 blood serum samples collected in August, antibodies against the hemagglutinin of H1, H3, H5, H6 and H9 subtypes of AIV were found in 1, 0, 2, 0 and 8 samples, respectively; and 11 samples were found with antibody against NDV. Whereas the NDV isolation in both two batches of specimens of mallard was negative, all of the 32 blood serum samples collected in December were negative for antibodies against AIV and NDV.

[Surgical approach in plastic of shortened lower limb].

OBJECTIVE: To introduce a new surgical approach to rectify the shortened lower limbs. METHODS: From March 1985 to October 2000, 288 cases of shortened lower limbs were treated and reviewed. Closed fracture at the metaphysis was made by a self-made "needle saw", and then the "multiple-plane and double-track elongation instrument" was adopted to elongate the fractured bone. There were totally 161 cases of male and 127 cases of female included, with average age 21.3 years old, ranging from 12 to 29 years old, among which there were 268 cases elongated at the proximal metaphysis of the tibia, 16 cases at the distal femur and 4 cases at the distal tibia. All of the cases were followed up for 6 to 8 months before clinical evaluation. RESULTS: The lower limbs in 288 cases were elongated for 3.0 to 11.5 cm in 24 to 96 days, averaging 47 days, which fulfilled pre-operative plan. In the second week after the operation, new calculus and periosteum formed obviously in the gap between the fractured parts, and in 6 to 8 months bone union was observed at the fractured site in all cases. There was no nerve or blood vessel injury, or non-union of the metaphysis fracture. The function of the manipulated knee joints and ankle joints recovered well. CONCLUSION: It is a practical and safe surgical option to rectify the shortened lower limbs by closed fracture at the metaphysis, followed by elongation of the fractured bone, without any complication such as non-union or atrophy of manipulated bone, and with no need of internal fixation or bone grafting.