Melatonin promoted osteogenesis of human periodontal ligament cells by regulating mitochondrial functions through the translocase of the outer mitochondrial membrane 20.

BACKGROUND AND OBJECTIVE: Melatonin plays an important role in various beneficial functions, including promoting differentiation. However, effects on osteogenic differentiation, especially in human periodontal cells (hPDLCs), still remain inconclusive. Mitochondria are highly dynamic organelles that play an important role in various biological processes in cells, including energy metabolism and oxidative stress reaction. Furthermore, the translocase of the outer mitochondrial membrane 20 (TOM20) is responsible for recognizing and transporting precursor proteins. Thus, the objective of this study was to evaluate the functionality of melatonin on osteogenesis in human periodontal cells and to explore the involved mechanism of mitochondria. METHODS: The hPDLCs were extracted and identified by flow cytometry and multilineage differentiation. We divided hPDLCs into control group, osteogenic induction group, and osteogenesis with melatonin treatment group (100, 10, and 1 µM). Then we used a specific siRNA to achieve interference of TOM20. Alizarin red and Alkaline phosphatase staining and activity assays were performed to evaluate osteogenic differentiation. Osteogenesis-related genes and proteins were measured by qPCR and western blot. Mitochondrial functions were tested using ATP, NAD+/NADH, JC-1, and Seahorse Mito Stress Test kits. Finally, TOM20 and mitochondrial dynamics-related molecules expression were also assessed by qPCR and western blot. RESULTS: Our results showed that melatonin-treated hPDLCs had higher calcification and ALP activity as well as upregulated OCN and Runx2 expression at mRNA and protein levels, which was the most obvious in 1 µM melatonin-treated group. Meanwhile, melatonin supplement elevated intracellular ATP production and mitochondrial membrane potential by increasing mitochondrial oxidative metabolism, hence causing a lower NAD+ /NADH ratio. In addition, we also found that melatonin treatment raised TOM20 level and osteogenesis and mitochondrial functions were both suppressed after knocking down TOM20. CONCLUSION: We found that melatonin promoted osteogenesis of hPDLCs and 1 µM melatonin had the most remarkable effect. Melatonin treatment can reinforce mitochondrial functions by upregulating TOM20.

[Pineal mechanism of danggui shaoyao powder on anti-aging].

OBJECTIVE: To study whether Danggui Shaoyao Powder (DSP) is by way of improving pineal function to realize its anti-aging effects. METHODS: Forty aged rats were randomly divided into the sham operated group (A), the pinealectomized group (B), the sham medicated group (C), the pinealectomized and medicated group (D). The medication given was gastric perfusion of DSP for 3 weeks. Learning and memory ability of rats was observed using Morris water maze and the serum melatonin (MLT) concentration of the rats was measured by radio-immunoassay. RESULTS: The average escape latency in Group B was significantly longer than that in other groups (P <0.05). The times of passing through the platform and the percentage of swimming distance in Group C were significantly higher than those in other groups (P <0.05). The serum MLT was higher at daytime than at night in Group C and D (P <0.05); that at daytime in Group C was higher than Group A (P <0.05) and also higher in Group D than Group B; that at daytime was higher in Group C than Group D; that at night markedly decreased in Group D as compared with Group B (P <0.05). CONCLUSION: DSP could increase the melatonin secretion and improve learning and memory ability. Since its effects reduced after pinealectomy, it could be deduced that improving pineal function should be one of the action mechanisms for anti-aging.

Effects of the aqueous extract of the Chinese medicine Danggui-Shaoyao-San on rat pineal melatonin synthesis.

OBJECTIVES: The purpose of this study is to investigate if the aqueous extract of the Chinese medicine Danggui-Shaoyao-San (DSS) can increase the plasma level of melatonin and enhance the function of the pineal gland of naturally aged rats. METHODS: The rats were treated with DSS at doses of 3ml or same volume of distilled water by oral administration at 11 p.m. for three weeks. The plasma level of melatonin were measured by radioimmunoassay. The function of pineal gland were measured through three parameters: pineal beta adrenergic receptor binding investigated by [3H]DHA binding; pineal expression of NAT mRNA detected by real-time RT-PCR; phosphorylation of CREB (P-CREB) and total level of CREB (T-CREB) measured by western blot analysis. RESULTS: DSS significantly increased melatonin level at night after oral administration for 3 weeks. By measurement of pineal [3H]DHA binding, it was found DSS improved the beta-adrenergic receptors binding in pineals. The stimulatory effect of DSS on the expression of NAT mRNA in the old rat pineal gland has been demonstrated in this study. Western blot analysis showed that DSS significantly increased phosphorylation of CREB. CONCLUSIONS: Our results indicate that a downstream pathway for DSS induction of melatonin synthesis in the rat pineal gland acts via cyclic AMP-dependent cascade and transcription mechanism.

Differential effects of melatonin on hippocampal neurodegeneration in different aged accelerated senescence prone mouse-8.

OBJECTIVES: A purpose of this study is to compare the differential effects of melatonin on hippocampal neurodegeneration in accelerated senescence prone mouse-8 (SAMP8) which is initiated treatment at different age. METHODS: The 4-months old SAMP8 mice were injected subcutaneously with melatonin (1 mg/kg/day) for 4 months. Similar treatments were performed in the 7-months old mice. When the animals were complete 11-months old, a series of tests were performed. Y maze test and Eight-arm radial maze task were used to assess cognitive performance. Hippocampal pyramidal cells were estimated by Nissl's staining. By using Gomori's methenamine silver methods, the methenamine silver staining granules (MSSG) were observed in area CA1 of hippocampus. A computer-assisted morphometric study was carried out on the ultrastructure of perikaryal CA1 pyramidal cell mitochondria. The volume density (Vv), surface density (Sv), numerical density (Nv) and mean volume (V) of the mitochondria were calculated. RESULTS: Melatonin treatment obviously reduced the deposition of MSSG and elevated hippocampal pyramidal cell number while improving the learning and memory deficits of SAMP8. The mice initiated treatment from 4-months old exhibited a greater response to melatonin supplementation than 7-months old mice. It also decreased mean volume (V) and significantly elevated the Sv and Nv of the mitochondria in hippocampal CA1 region. However, 7-months old mice showed little effects on it. CONCLUSIONS: Our results indicate that the protective effects of melatonin on hippocampal neurodegeneration of SAMP8 are age dependent.

[Effects of dahuang zhechong pill on expression and activity of matrix metalloproteinase in rats' hepatic stellate cells].

OBJECTIVE: To observe the effects of Dahuang Zhechong Pill (DZP) on the gene expression of matrix metalloproteinase-1 (MMP-1) in rats' hepatic stellate cells (HSC) and activity of matrix metalloproteinase-2 (MMP-2) that secreted into the culture base. METHODS: HSC were isolated from the liver of normal rats and incubated with DZP-contained drug serum. The expression of MMP-1 in the HSC was detected by quantitative reverse-transcription polymerase chain reaction (RT-PCR). The activity of MMP-2 was examined by zymography. RESULTS: DZP-contained drug serum could obviously promote the gene expression of MMP-1 in HSC. In the meantime, it could obviously increase the content and activity of MMP-2 synthesized by HSC (P < 0.05). CONCLUSION: The anti-fibrosis action of DZP was correlated to the promotion of HSC's gene expression of MMP-1 and increasing of the contents and activity of MMP-2.

[Effect of supplemented danggui shaoyao powder and its disassembled prescriptions on lymphocytes proliferation in aged rats].

OBJECTIVE: To study the effect of supplemented Danggui Shaoyao San (DSS) and its disassembled prescriptions on function of lymphocyte in aged rats. METHODS: Rats aged 18 months were administered with decoction of DSS and its disassembled prescriptions separately in groups for 3 weeks to prepare drug containing serum. Samples of ConA induced rats splenic lymphocytes proliferation was treated respectively with prepared drug serum, for testing their effect on lymphocyte proliferation using methyl thiazolyl tetrazolium (MTT) method, and isoprel (ISO) and propranolol were taken as the controls. RESULTS: The A value of lymphocyte proliferation in the drug serum treated groups was significantly different from that in the control groups (P < 0.01), which could be reduced markedly after treatment with ISO, but could restore to the level before ISO treatment by adding drug serum or propranolol. CONCLUSION: DSS and/or its disassembled prescriptions could raise the lymphocyte proliferation in aged rats significantly, it also shows antagonizing effect against the inhibition of ISO on lymphocyte proliferation.