ObjectiveTo explore the effects of heat treatment and ultraviolet B (UVB) radiation alone or in combination on the expression of heat shock protein (HSP) 72 in human epidermal melanocytes.Methods Melanocytes were obtained from human foreskin,and subjected to primary culture.After 3 to 5 passages,the melanocytes were classified into 4 groups:control group (receiving no treatment),heat treatment group (treated with heat at 42 ℃ for 1 hour every day for 3 days),UVB group(irradiated with UVB at 50 mJ/cm2 daily for 3days),combination group(treated with heat at 42 ℃ for 1 hour followed by irradiation with UVB at 50 mJ/cm2daily for 3 days).After another 2- to 6-hour culture following the last treatment,melanocytes were collected and subjected to real time PCR and Western blot for the detection of HSP72 mRNA and protein expression,respectively.ResultsThe mRNA and protein expressions of HSP72 were significantly higher in the heat treatment group and combination group than in the control group (mRNA:6.584 ± 0.871 and 7.269 ± 0.454 vs.0.975 ± 0.089,both P ＜ 0.001; protein:2.022 ± 0.058 and 2.080 ± 0.045 vs.0.532 ± 0.033,both P ＜ 0.001 ),but was similar between the UVB group and control group (mRNA:0.832 ± 0.084 vs.0.975 ± 0.089,P ＞ 0.05;protein:0.546±0.021 vs.0.532 ± 0.033,P ＞ 0.05).The ANOVA of factorial design showed that neither heat treatment nor UVB irradiation had interaction effect on the mRNA or protein expression of HSP72 (F =2.106,1.399 respectively,both P ＜ 0.05).ConclusionsHeat treatment can cause an increase in the expression of HSP72,which may enhance the function of melanocytes and protect melanocytes from UVB induced damage.

ObjectiveTo investigate the effect of heat treatment combined with narrow band ultraviolet B(NB-UVB) on cultured normal human melanocytes in vitro.MethodsMelanocytes were isolated from the foreskin of normal human,cullured in vitro,and irradiated with NB-UVB of different doses(20,30,50,70,90,120 and 180 mJ/cm2).Then,MTT assay was performed to evaluate the proliferation and activity of melanocytes to determine the optimal dose of UVB for the next experiment.Melanocytes were classified into 3 groups to be treated with heat at 42 ℃ for 1 hour (heat group),irradiated with UVB at 50 mJ/cm2 (UVB group),or irradiated with UVB at 50 mJ/cm2 followed by heat treatment at 42 ℃ for 1 hour (combination group),daily for 3 successive days; those receiving no treatment served as the control.After 24-hour culture following the last treatment,tyrosinase activity was evaluated with L-dopa as the substrate,melanin content was detected by NaOH assay,and cell cycle stages were determined by flow cytometry.ResultsNB-UVB irradiation decreased the viability of melanocytes in a dose-dependent manner,and the optimum dose of UVB was 50 mJ/cm2.The tyrosinase activity of melanocytes was 0.244 ± 0.018 and 0.310 ± 0.015 respectively in the UVB group and combination group,and increased by 3.8％ (P ＜ 0.05) and 31.9％ (P ＜ 0.05) respectively compared with the control group (0.235 ± 0.018); the melanin content was 0.201 ± 0.016 and 0.286 ± 0.019,respectively in the UVB group and combination group,and increased by 17.5％ (P ＜ 0.05 ) and 67.3％ (P ＜ 0.05) compared with the control group (0.171 ± 0.016).In comparison with the control group,the percentage of melanocytes in G1 phase was decreased by 23.94％ in the UVB group(P＜ 0.05) and 33.51％ in the combination group(P ＜ 0.05),while that in S phase and G2 phase increased by 15.35％ (P ＜ 0.05 ) and 11.93％ (P ＜ 0.05),respectively in the UVB group,and 17.76％ (P ＞ 0.05) and 16.08％ (P ＞ 0.05),respectively in the heat group.ConclusionHeat treatment and NB-UVB can synergistically enhance the tyrosinase activity and accelerate melanogenesis,proliferation and differentiation,of melanocytes.

Objective To investigate the mRNA and protein expressions of nine integrin subunits in human keloid-derived mesenchymal stem cells (KD-MSCs).Methods Cultured KD-MSCs and normal skin-derived MSCs (NS-MSCs) served as the experiment group and control group respectively.Real-time quantitative PCR and Western blot were performed to measure the mRNA and protein expressions of nine integrin subunits in the two groups respectively.Statistical analysis was carried out by t test.Results Real-time quantitative PCR showed no significant difference in the mRNA expression level of any of the integrin units α2,α3,α5,αV,α10,α11 or β1 between KD-MSCs and NS-MSCs (all P ＞ 0.05).The mRNA expression level of integrin α8 was decreased,while that of integrin β3 was significantly increased in KD-MSCs compared with NS-MSCs (both P ＜ 0.01).Western blot revealed that the changes in protein expression levels of integrin units α8 and β3 were consistent with those in their mRNA expression levels in both KDMSCs and NS-MSCs (both P ＜ 0.01).Conclusions Integrin units α8 and β3 may be involved in the occurrence and development of keloid,and the receptors made up of them may play important roles in the pathogenesis of keloid.

Objective To investigate the action mechanism of glutamate-mediated signaling pathway in malignant melanoma. Methods WM451LU melanoma cells in log phase were classified into 6 groups, negative control group treated with PBS (100 μl), MK801 group treated with the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (100 μmol/L), CPCCOEt group treated with non-competitive metabotropic glutamate receptor 1 (mGluR1) antagonist CPCCOEt, MAP2 group transfected with adenovirus vector containing microtubule associated protein 2a (Ad-MAP2a), MK801 + MAP2 group treated with MK801 of 100 μmol/L and transfected with Ad-MAP2a, CPCCOEt + MAP2 group treated with CPCCOEt of 10 μmol/L and transfected with Ad-MAP2a. Western blot was performed to detect the expression of an ionotropic glutamate receptor, i.e., N-methyl-D-aspartate receptor type 2A (NMDAR2A) in WM451LU cells transfected with Ad-MAP2a. Scratch motility assay and cell invasion assay were conducted in vitro to detect the changes in migration and invasion ability of WM451LU cells after treated with Ad-MAP2a, MK-801, CPCCOEt alone or in combination. In vivo study was carried out to compare the inhibitory effect of the above treatments on melanoma. Results Western blot revealed a decrease in the expression of NMDAR2A in WM451LU cells after transfected with Ad-MAP2a. The scratch motility assay showed that the number of migrating cells per high power field was 117.04 ± 2.76 in MAP2 group,107.64 ± 6.50 in MK801 group,97.36 ± 4.79 in CPCCOEt group, 43.28 ± 3.02 in MK801 + MAP2 group,30.76 ± 3.97 in CPCCOEt + MAP2 group,significantly different from that in the negative control group (152.3 ± 5.75,all P ＜ 0.01 ). Cell invasion assay demonstrated that the average number of invading cells per high power field in the negative control was significantly higher than that in MAP2 group, MK801 group, CPCCOEt group, MK801+MAP2 group and CPCCOEt + MAP2 group (170.43 ±8.72 vs. 98.26 ± 3.84, 97.22 ± 5.54, 112.23 ± 7.21, 42.89 ± 5.06, 58.25 ± 6.68, P ＜ 0.05, 0.05, 0.05, 0.01and 0.01, respectively).A significant decrease was observed in the average volume of experimental melanoma in mice of MAP2 group, MK801 group, MK801 + MAP2 group, CPCCOEt group and CPCCOEt + MAP2 group compared with the negative control group (224.02 ± 46.19 mm3, 160.33 ± 33.91 mm3, 91.49 ± 21.48 mm3,202.30 ± 52.37 mm3, 111.13 ± 69.81 mm3 vs. 342.70 ± 60.92 mm3, all P ＜ 0.01 ). Conclusions To block the glutamate signaling pathway in vitro can inhibit the invasion and migration of melanoma cells, and to block the pathway in vivo can inhibit the growth of malignant melanoma and alter the morphology of melanoma cells.

ObjectiveTo observe the increasing effect of infrared irradiation on tyrosinase activity and melanin content in cultured normal human epidermal melanocytes in vitro and to explore the optimal dose of infrared irradiation.MethodsEpidermal melanocytes were isolated from normal human foreskin tissue,and subjected to primary culture.Methyl thiazolyl tetrazolium(MTT) assay was performed to evaluate the effect of different doses(0,20,60,80,100,140,240,320 J/cm2)of infrared light on the proliferation of melanocytes and to select the optimal irradiation dose.Then,melanocytes were irradiated with infrared light at the optimal dose for 3 consecutive days followed by the determination of tyrosinase activity,melanin content,and cell cycle via dopa oxidation assay,NaOH solubilization method and flow cytometry,respectively.ResultsThe best intervention dose of infrared light was 80 J/cm2.The tyrosinase activity(A492 nm) and melanin content(A492 nm)were 0.3601 ± 0.0301 and 0.2748 ± 0.0243 respectively in melanocytes after irradiation with infrared light of 80 J/cm2 for 3 days,significantly higher than those in unirradiated melanocytes(0.3114 ± 0.0341,0.2325 ±0.0254,respectively,both P ＜ 0.05),with an increase rate of 15.64％ and 18.19％ respectively.Cell cycle analysis revealed a decline in cell percentage in G1 phase(P ＜ 0.01 ) but a concomitant increase in cell percentage in G2 and S phase (both P ＜ 0.05) in irradiated melanocytes compared with unirradiated melanocytes.ConclusionsThe optimal dose of infrared light is 80 J/cm2 for the irradiation of melanocytes,and this dose of infrared light can increase melanin content,tyrosinase activity,differentiation and proliferation of melanocytes.

This study was aimed to observe the body weight and behavioral changes of heart-spleen deficiency mouseandtoassesstheefficacyofLing-Qi-Jia (LQJ)oralsolutiononqi-supplementingandmind-tranquilizing. The heart-spleen deficiency syndrome mouse model was established by using loading swimming anddrugdaily.Thebodyweight,foodconsumption,intestinepropulsion,tailsuspensiontest (TST),forced swimmingtest (FST),sleepingtimeandtheamountofbrainneurotransmitterweredetected.Theresultsshowed that mouse suffered loading swimming and drug formed heart-spleen deficiency syndrome model, which were indicated by lowering body weight and food consumption, shortened time in FST, prolonged accumulative immobility time in TST, intestine propulsion hyperfunction, shortened sleeping time and lowering brain neurotransmitter amount. LQJ oral solution can obviously improve experiment indexes mentioned above. It was concluded that LQJ oral solution, which can improve insomnia due to heart-spleen deficiency, might had close relation to the efficacy of qi-supplementing and mind-tranquilizing. Meanwhile, changes of brain neurotransmitters might also be the material basis on its efficacy.

This study was aimed to observe the effect ofRong-Shuan (RS) capsule on rodent tolerance against cerebral ischemia, hypoxia and cerebral reserve capacity, which was related to promote blood circulation and remove blood stasis. Acute cerebral ischemia and anoxia models were established by permanent left carotid artery ligation on C57 BL/6 mice and hypoxia inhalation (O2?N2 = 8?92) for 15 min. Duodenal administration of RS capsule at different doses (100, 200 or 400 mg·kg-1) or saline were given 10 min after ischemia onset. The local brain blood circulation changes and neurobehavioral function were evaluated 24 h after ischemia onset. SD rats were given RS capsule at different doses (75, 150, 300 mg·kg-1) or saline. The effect of RS capsule on improvement of microcirculation disturbance induced by high molecular dextran was observed. The results showed that compared with the sham operation group, the brain blood circulation in the model group was significantly decreased; the cerebral infarction area increased; and the behavioral score after cerebral hypoxia was significantly increased (P < 0.05, orP < 0.01). Meanwhile, after the injection of high molecular dextran among rats in the model group, the cerebral leptomeninx microcirculation was obviously slowed down at 3 timepoints, which were 10, 20 and 30 min. Compared with the model group, RS capsule (400 mg·kg-1) can significantly increase the local blood circulation in the brain of mice, improve behavioral disturbance, reduce cerebral ischemia area (P< 0.05, orP < 0.01). RS capsule can also improve blood flow velocity and flow pattern in cerebral leptomeninx microcirculation disturbance induced by high molecular dextran at different timepoints (P < 0.05, orP < 0.01). It was concluded that RS capsule can increase the tolerance against cerebral ischemia, hypoxia and cerebral reserve capacity in order to protect the neural tissues to promote neuronal recovery.

This study was aimed to observe behavioral changes of post-stroke depression (PSD) rats, and to assess effect ofJie-Du Tong-Luo(JDTL) capsule onqi-supplementing and depression-relieving. Microspheres were injected from external carotid artery of rats under anesthesia to prepare the multiple cerebral infarction. Aftermid long term feeding, PSD rat model was established. Then, open-field test (OFT), tail suspension test (TST), forced swimming test (FST) and glucose preference test were employed to study behavioral changes of rats. The results showed that rats suffered multiple cerebral infarction after mid long term feeding formed PSD, which were indicated by reduced food consume, slow body weight increasing, reduction of spontaneous movement and inquiry activity, prolonged accumulative immobility time in TST, FST and lowered glucose preference, compared with rats in the normal group. Compared with the model group, rats in the JDTL capsule group andBu-Chang Xin-Nao-Tonggroup showed larger body weight increase, higher scores in OFT, reduced immobility time in TST, FST, and elevated glucose preference. It was concluded that JDTL capsule had significant efficacy on rats’ body weight, behavior and glucose preference, which might be its pharmacological basis onqi-supplementing and depression-relieving.

The rat model of multi-infarct was adopted in this study to elucidate the protective mechanism of Sailuotong capsule (Sailuotong) in recovery period of multiple cerebral infarction. The effects of Sailuotong on levels of Glu, GABA and the expression of NMDA receptor subtypes including NR1, NR2A and NR2B, were detected. The multi-infarct model rats were established by injecting embolizing microsphere via internal carotid artery, and were given Sailuotong treatment (16.5 and 33.0 mg x kg(-1)) for 60 days. The pathological changes in brain ultrastructure were observed by transmission electron microscope. The levels of Glu and GABA in brain tissue were measured with high performance liquid chromatography. The expression of NMDA receptors including NR1, NR2A and NR2B in neurons was evaluated by immunohistochemical staining. Compared with the sham rats, abnormal changes were observed in ultrastructures of neurons, neuroglia cells and synapses of model rat brains. Moreover, significant decrease of Glu and GABA, as well as the elevated expression of NR1, NR2A and NR2B were detected in brain tissues. Sailuotong (16.5 and 33.0 mg x kg(-1)) could improve ultrastructure of cerebral tissue, facilitate synthesis of Glu and GABA, and down-regulate expression of NR1, NR2A and NR2B in neurons. The results demonstrated that Sailuotong could exert neuroprotective effects to some extent in the recovery phase of multiple cerebral infarction by promoting expression of NMDA receptors and synthesis of Glu and GABA.

Recent publications are quoted to summarize multiple use of microdialysis in medical fields, especially in pharmacology and pharmacokinetics. Microdialysis was coupled with HPLC-ECD, HPLC-MS and other detectors to study endogenous substances and medicines, including neurotransmitters, amino acid, other endogenous metabolites and drugs as well as Chinese medicines. Microdialysis is a relatively new sampling technique and its advantages as well as disadvantages are briefly assessed. At the end of this review, an outlook to apply this technique in traditional Chinese medicine study is given forward.
Amino Acids ; analysis ; Chromatography, High Pressure Liquid ; methods ; Humans ; Medicine, Chinese Traditional ; methods ; Microdialysis ; methods ; trends ; Neurotransmitter Agents ; analysis ; Pharmaceutical Preparations ; analysis