Increasing evidence suggests that stress may induce changes in hair color, with the underlying mechanism incompletely understood. In this study, female C57BL/6 mice subjected to electric foot shock combined with restraint stress were used to build chronic stress mouse model. The melanin contents and tyrosinase activity were measured in mouse skin and B16F10 melanoma cells. The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of tumor necrosis factor α (TNF-α), interleukin- 1β (IL-1β) and interleukin-6 (IL-6) in the mouse skin. The content of nuclear factor κB (NFκB)/p65 subunit in mouse skins was valued by immunofluorescence staining. The results demonstrated that under chronic stress, the fur color turned from dark to brown in C57BL/6 mice due to the decrease of follicle melanocytes and tyrosinase activity in C57BL/6 mouse skin. Simultaneously, inflammatory responses in skins were detected as shown by increased NFκB activity and TNF-α expression in stressed mouse skin. In cultured B16F10 melanoma cells, TNF-α reduced the melanogenesis and tyrosinase activity in a dose-dependent manner. These findings indicate that chronic stress induces fur color change by decreasing follicle melanocytes and tyrosinase activity in female C57BL/6 mice, and TNF-α may play an important role in stress-induced hair color change.
Animal Fur ; Animals ; Color ; Female ; Melanins ; Melanocytes ; enzymology ; Melanoma, Experimental ; Mice ; Mice, Inbred C57BL ; Monophenol Monooxygenase ; metabolism ; Pigmentation ; Skin ; physiopathology ; Stress, Physiological

OBJECTIVETo investigate the role of integrin-linked kinase (ILK) signaling pathway in the skin lesions and wound healing in diabetic rats.

METHODSThirty-six SD rats were divided into diabetic wound group (D) and non-diabetic wound group (N) according to the random number table, with 18 rats in each group. 10 g/L streptozocin (60 mg/kg) was intraperitoneally injected in rats in group D, while the rats in group N were given same quantity of sodium citrate buffer. Two weeks after successful reproduction of diabetic model of rats in group D, two full-thickness skin of an area of 2 cm × 2 cm was resected on both sides of back of rats in the two groups. Wounds of three rats of each group were photographed and examined on post injury day (PID) 1, 3, 7, 10, 14, and 21, and the wound healing rates were calculated. The non-injured skin and wound tissue (central part) on back of three rats of the rest 15 rats in the two groups were harvested on PID 3, 7, 10, 14, and 21, respectively. Morphology of the non-injured skin tissue was observed with HE staining, and the thickness of full-thickness skin and epidermis were measured. The mRNA expression levels of ILK, protein kinase B (Akt), and glycogen synthase kinase-3β (GSK-3β) in non-injured skin tissue were determined with real-time fluorescent quantitative RT-PCR. The protein expression levels of ILK, Akt, phosphorylated Akt, GSK-3β, and phosphorylated GSK-3β in non-injured skin tissue, and ILK, phosphorylated Akt in wound tissue were assessed with Western blotting. Data were processed with two independent-sample t test, one-way analysis of variance, SNK test and analysis of variance of factorial design.

RESULTS(1) After injury, the wound scabs of rats in group N were dry, and red granulation tissue with no excretion were seen when the scabs fell off, and the wound healed fast. After injury, excretion under the wound scabs of rats in group D was seen, and the scabs easily fell off with exposure of pink granulation tissue with much excretion, and the wounds healed slowly. Except for PID 3, the wound healing rate of rats in group D was significantly lower than that in group N on other PIDs (with t values from 3.858 to 13.738, P<0.05 or P<0.01). (2) On PID 3, the hair follicles and blood vessels in the non-injured skin tissue of rats in group N were rich, and the epidermis was composed of stratified cells in form of basal cells and keratinocyte, and the hair follicles and blood vessels in the non-injured skin tissue of rats in group D were scarce, and the epidermis was nearly composed of one-layer of cells. The thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group N was similar from PID 3 to 21, and the thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group D on PID 3 was respectively (1 074 ± 66) and (15.1 ± 3.8) μm, and they gradually thinned out to (785 ± 122) and (9.7 ± 2.1) μm on PID 21, respectively. The thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group N were significantly thicker than those in group D on each PID (with t values from 4.620 to 23.549, P values below 0.001). (3) From PID 3 to 21, the mRNA expression levels of ILK and Akt in non-injured skin tissue of rats in group D were significantly lower than those in group N (with t values respectively 4.779 and 3.440, P values below 0.05), the mRNA expression levels of GSK-3β in non-injured skin tissue of rats were similar in two groups (t=0.363, P>0.05). (4) From PID 3 to 21, the protein expression levels of ILK, Akt and phosphorylated Akt in non-injured skin tissue of rats in group D were significantly lower than those in group N (with t values from 2.630 to 6.209, P<0.05 or P<0.01); the protein expression levels of GSK-3β in non-injured skin tissue of rats in two groups were similar (t=0.652, P>0.05); the protein expression level of phosphorylated GSK-3β in non-injured skin tissue of rats in group D was significantly higher than that in group N (t=4.131, P<0.001). The protein expression levels of ILK in wound tissue of rats in two groups were similar on each PID (with t values from 0.381 to 2.440, P values above 0.05). Except for PID 3, the protein expression levels of phosphorylated Akt in wound tissue of rats in group N were significantly higher than that in group D on other PIDs (with t values from 4.091 to 20.555, P<0.05 or P<0.01). From PID 3 to 21, the protein expression levels of ILK in wound tissue and non-injured skin tissue of rats in group N were similar (F=2.522, P>0.05), and the protein expression level of phosphorylated Akt in wound tissue was significantly higher than that in non-injured skin tissue (F=117.329, P<0.001); the protein expression levels of ILK in wound tissue and non-injured skin tissue of rats in group D were similar (F=1.337, P>0.05), and the protein expression level of phosphorylated Akt in wound tissue was significantly higher than that in non-injured skin tissue (F=184.120, P<0.001).

CONCLUSIONSThe skin lesion of diabetic rats may be related to the declined expression levels of ILK, Akt and phosphorylated Akt in the ILK signaling pathway. The refractory healing of wound in diabetic rats may be related to the declined expression level of phosphorylated Akt.

Animals ; Diabetes Mellitus, Experimental ; enzymology ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Phosphorylation ; Protein-Serine-Threonine Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Skin ; injuries ; Wound Healing

OBJECTIVETo investigate the role of extracellular signal-regulated kinase1/2 (ERK1/2) pathway in the regulation of aquaporin 4 (AQP4) expression in cultured astrocytes after scratch-injury.

METHODSThe scratch-injury model was produced in cultured astrocytes of rat by a 10-μL plastic pipette tip. The morphological changes of astrocytes and lactate dehydrogenase (LDH) leakages were observed to assess the degree of scratch-injury. AQP4 expression was detected by immunofluorescence staining and Western blot, and phosphorylated-ERK1/2 (p-ERK1/2) expression was determined by Western blot. To explore the effect of ERK1/2 pathway on AQP4 expression in scratch-injured astrocytes, 10 µmol/L U0126 (ERK1/2 inhibitor) was incubated in the medium at 30 min before the scratch-injury in some groups.

RESULTSIncreases in LDH leakage were observed at 1, 12, and 24 h after scratch-injury, and AQP4 expression was reduced simultaneously. Decrease in AQP4 expression was associated with a significant increase in ERK1/2 activation. Furthermore, pretreatment with U0126 blocked both ERK1/2 activation and decrease in AQP4 expression induced by scratch-injury.

CONCLUSIONThese results indicate that ERK1/2 pathway down-regulates AQP4 expression in scratch-injured astrocytes, and ERK1/2 pathway might be a novel therapeutic target in reversing the effects of astrocytes that contribute to traumatic brain edema.

Animals ; Aquaporin 4 ; metabolism ; Astrocytes ; enzymology ; metabolism ; Butadienes ; administration & dosage ; Cells, Cultured ; Down-Regulation ; Enzyme Activation ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; MAP Kinase Signaling System ; Nitriles ; administration & dosage ; Rats ; Rats, Wistar ; Skin ; injuries

Cell Line, Tumor ; Humans ; Matrix Metalloproteinase 2 ; analysis ; Matrix Metalloproteinase 9 ; analysis ; Melanoma ; enzymology ; Skin Neoplasms ; enzymology

OBJECTIVETo study the effects of Zhuhong ointment on accumulation in the body of mercury and the pathological morphology changes of kidney, via the measurement of related indicators of the skin-impaired model rat.

METHODEighty-eight SD rats were randomly divided into the impairment control group, and high-, middle-, low-dose Zhuhong ointment groups. Each group was treated by corresponding methods for 4 weeks, and recovering for 4 weeks. Urinary potein (PRO), pH, Beta N-acetyl aminoglycosidase enzymes (NAG) and beta2-microglobulin (beta2-MG) contents in urine were taken as monitoring indexes, blood urea nitrogen (BUN) and serum creatinine (SCr) in blood and the levels of mercury in urine, blood and kidney were tested, and the pathological morphology changes of kidney were observed.

RESULTAfter treatment for 4 weeks, compared with impairment control group, the levels of mercury in urine, blood and kidney in every dose group increased significantly (P < 0.01). And the relation exists between toxicity and dose on Zhuhong ointment. After recovery for 4 weeks, the levels of mercury in urine and blood in every dose group restore normal, while the level of mercury in kidney in high- dose group still increased (P < 0.01). The level of NAG increased only in high-dose group. There was no significant difference in NAG contents between Zhuhong ointment groups and the impairment control group (P < 0.05).

CONCLUSIONExcess using Zhuhong ointment repeatedly may lead to accumulation of mercury and pathological morphology changes of kidney. So the levels of mercury in the body and related indicators of renal functions should be tested in clinical when long-term using Zhuhong ointment.

Acetylglucosaminidase ; drug effects ; urine ; Animals ; Blood Urea Nitrogen ; Creatinine ; blood ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; toxicity ; Female ; Hydrogen-Ion Concentration ; drug effects ; Kidney ; drug effects ; enzymology ; metabolism ; pathology ; Male ; Mercury ; blood ; metabolism ; urine ; Ointments ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Retinol-Binding Proteins ; drug effects ; urine ; Skin ; drug effects ; injuries ; Time Factors ; beta 2-Microglobulin ; urine

Animals ; Carcinoma, Squamous Cell ; genetics ; Colorectal Neoplasms ; genetics ; metabolism ; Enzyme Activation ; Esophageal Neoplasms ; genetics ; Genome-Wide Association Study ; Head and Neck Neoplasms ; genetics ; Humans ; Neoplasms ; chemically induced ; enzymology ; genetics ; Phosphoinositide Phospholipase C ; chemistry ; genetics ; metabolism ; physiology ; Signal Transduction ; Skin Neoplasms ; chemically induced ; enzymology ; Stomach Neoplasms ; genetics ; Urinary Bladder Neoplasms ; metabolism ; pathology ; ras Proteins ; metabolism

OBJECTIVETo study the effection of suppression murine melanoma growth by Intratumor injection of recombinant attenuated salmonella carrying heat shock protein 70 and herpes simplex virus thymidine kinase genes.

METHODSPlasmids PCMV-mtHSP70-IRES-TK were electro-transferred into salmonella typhimurium SL7207 to construct recombinant salmonella typhimurium. In vivo, Recombinant bacteria were injected into the mouse melanoma and the antitumor effection was observed. The survival period was recorded and safety analysis for this vaccine in each group.

RESULTSIn vivo, the mtHSP70/HSV-tk recombinant bacteria can suppress tumor growth significantly and extend survival. After recombinant Salmonella, 10(9) CFU/mL, was administered as an intratumoral injection, No diarrhea were observed. During therapy, body weight did not change markedly.

CONCLUSIONResults of the animal experiment suggests intratumor injection of recombinant attenuated salmonella typhimurium containing mtHSP70 and HSV-tk genes, has targeting ability against B16 tumor cell and could significantly inhibit tumor growth .

Animals ; Bacterial Proteins ; genetics ; immunology ; Cancer Vaccines ; genetics ; immunology ; pharmacology ; Genetic Therapy ; methods ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Melanoma, Experimental ; microbiology ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Mycobacterium tuberculosis ; genetics ; Salmonella typhimurium ; genetics ; immunology ; Simplexvirus ; enzymology ; genetics ; Skin Neoplasms ; therapy ; Thymidine Kinase ; genetics ; immunology ; Vaccines, Attenuated ; genetics ; immunology ; pharmacology ; Vaccines, DNA ; genetics ; immunology ; pharmacology

OBJECTIVE: To assess the role of p38 in fibroblast orientation and to explore the cell signal transduction mechanism of cyclic strain induced cell orientation. METHODS: Fibroblasts were seeded onto collagen coated flexible membranes. Membranes were then deformed at 10 cycles per minute under 135 mmHg subatmospheric pressure. Orientation angles of cells treated with or without SB203580 were measured with inverted microscope. P38 phosporylation was analyzed with Western blot. RESULTS: Eighty percent cyclic strain induced cells rotated from 60 degree to 90 degree perpendicular to stretch direction after 4 h strain exposure. P38 phosphorylation reached the peak at 5 min. Fibroblast orientation was inhibited after SB203580 treatment. CONCLUSION Fibroblast orientation in response to cyclic strain is mediated by p38 phosporylation.
Cell Movement ; Cells, Cultured ; Enzyme Inhibitors ; pharmacology ; Fibroblasts ; cytology ; enzymology ; Humans ; Imidazoles ; pharmacology ; Male ; Mechanotransduction, Cellular ; physiology ; Phosphorylation ; Pyridines ; pharmacology ; Skin ; cytology ; enzymology ; Stress, Mechanical ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

Proteases in the skin are essential to epidermal permeability barrier homeostasis. In addition to their direct proteolytic effects, certain proteases signal to cells by activating protease-activated receptors (PARs), the G-protein-coupled receptors. The expression of functional PAR-2 on human skin and its role in inflammation, pruritus, and skin barrier homeostasis have been demonstrated. Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by genetic barrier defects and allergic inflammation, which is sustained by gene-environmental interactions. Recent studies have revealed aberrant expression and activation of serine proteases and PAR-2 in the lesional skin of AD patients. The imbalance between proteases and protease inhibitors associated with genetic defects in the protease/protease inhibitor encoding genes, increase in skin surface pH, and exposure to proteolytically active allergens contribute to this aberrant protease/PAR-2 signaling in AD. The increased protease activity in AD leads to abnormal desquamation, degradation of lipid-processing enzymes and antimicrobial peptides, and activation of primary cytokines, thereby leading to permeability barrier dysfunction, inflammation, and defects in the antimicrobial barrier. Moreover, up-regulated proteases stimulate PAR-2 in lesional skin of AD and lead to the production of cytokines and chemokines involved in inflammation and immune responses, itching sensation, and sustained epidermal barrier perturbation with easier allergen penetration. In addition, PAR-2 is an important sensor for exogenous danger molecules, such as exogenous proteases from various allergens, and plays an important role in AD pathogenesis. Together, these findings suggest that protease activity or PAR-2 may be a future target for therapeutic intervention for the treatment of AD.
Anti-Infective Agents/pharmacology ; Dermatitis, Atopic/*enzymology ; Endopeptidases/metabolism ; Homeostasis ; Humans ; Hydrogen-Ion Concentration ; Inflammation ; Models, Biological ; Models, Genetic ; Peptide Hydrolases/*metabolism ; Receptor, PAR-2/*metabolism ; Serine Proteases/metabolism ; Signal Transduction ; Skin/enzymology/pathology ; Treatment Outcome

OBJECTIVETo observe the effect of acupuncture on the expression of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in the subcutaneous fascia of SD rats.

METHODSEighteen SD rats were randomly divided into 6 groups (n=3) including 5 acupuncture groups and a control group. The rats in the 5 acupuncture groups received electro-acupuncture therapy in the regions of the inguinal groove, and at 0, 1, 6, 12, and 36 h after the last therapy, the superfacial fascia surrounding the acupuncture point (about 1.5 cm in diameter) were collected. The fascia tissues at the corresponding sites and at the acupoint Zusanli (ST36) were obtained from the control rats. The expression of ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in the tissues were detected by Western blotting.

RESULTSERK1/2 and p-ERK1/2 expressions were detected in the tissues harvested from both the acupoint and the non-acupoint in the control rats with similar expression intensities. In the rats of each acupuncture group, ERK1/2 expression was significantly increased on the acupuncture side in comparison with the control side.

CONCLUSIONThe normal loose connective tissue may participate in tissue proliferation and differentiation possibly via phosphorylation of ERK. Acupuncture can promote the signal transduction pathway of ERK, which can be a possible mechanism for the effect of acupuncture in modulating the physiopathological conditions.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Blotting, Western ; Fascia ; enzymology ; Female ; Gene Expression Regulation, Enzymologic ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Skin ; Time Factors