Experimental Biology and Medicine

Five decades after the first documented use of a laser for wound healing, research in light therapy has yet to elucidate the underlying biochemical pathways causing its effects. The aim of this review is to summarize the current research into the biochemical mechanisms of light therapy in order to better direct future studies. The implication of cytochrome c oxidase as the photoacceptor modulating light therapy is reviewed, as are the predominant hypotheses of the biochemical pathways involved in the stimulation of wound healing, cellular proliferation, production of transcription factors and other reported stimulatory effects.

A reliable animal model of facial nerve (FN) injury forms the basis for effective scientific studies of injury and repair of the FN. Currently, rodents are the most widely used animal model for such studies, most of which adopt a postauricular incision approach. However, it is difficult to carry out the procedure on perinatal rodents owing to characteristics of anatomy and the direction of incision. Based on anatomical studies on the extracranial segment of the FN in mice, we established a FN axotomy model by using a subauricular incision (SI) approach. The effect and the outcome of the FN transection were evaluated by electrophysiology, behavioral assessment and histological observation in 20 healthy four-week-old BALB/c mice. Favorable results were obtained in all of the mice and none died during the operation or subsequent observation period. Compared with the classic postauricular incision approach, the SI approach possesses multiple advantages including easier access, more satisfactory exposure of the FN trunk, minimal invasion and providing a larger operation field, which would be critical for those studies on the rodent in early development or delicate surgical manipulations.

The purpose of this study was to analyze the incidence, etiology and clinical characteristics of community-acquired pneumonia (CAP) among outpatients with sore throat and/or cough, and thus to provide theoretical basis for timely and accurate diagnosis and treatment for CAP. We used chest X-rays for fever (a temperature greater than 37.58C) patients, who were recruited since 2007, presenting with sore throat and/or cough. The patients’ age, gender, days of fever, respiratory symptoms (e.g. cough and sputum), peripheral blood count and etiology (pathogens) of CAP were recorded. Of all the 6539 fever outpatients, those aged 10-39 and above 60 years old accounted for 61.0% and 15.6%, respectively. In total, 402 were diagnosed with CAP with an incidence rate of 6.1%. Among them, 38.1% were above 60 years old. The prevalence increased with age. Of the 402 CAP patients, 36.8% (148/402) presented no respiratory symptoms and 30.1% (121/402) had positive etiology. The top three pathogens were Mycoplasma pneumoniae (23.1%), Streptococcus pneumoniae (17.3%) and Haemophilus influenzae (9.1%). Among the 121 etiology-positive patients, 14 cases were mixed infections (at least one atypical pathogen). Nine cases were M. pneumoniae mixed with bacterial/virus infection, and five cases were Chlamydia pneumoniae mixed with other bacteria/ mycoplasma infection. We found that fever outpatients have a higher prevalence of CAP, which increases with age, i.e. older people are more susceptible to CAP. S. pneumoniae and H. influenzae are common pathogens in CAP; however, atypical pathogens, especially M. pneumoniae, remain the most common pathogens in CAP.

The cholesterol-modulating, immune-regulating and anti-inflammatory properties of cordycepin are well documented. Here we examined the effects of triacetyl-3-hydroxyphenyladenosine (THPA), a derivative of cordycepin, on the development of atherosclerosis (AS) in apolipoprotein E-knockout (apoE^-/-) mice. The atherosclerotic lesion formation displayed by the oil red O staining-positive area was reduced significantly in either the aortic root section or the whole aorta en face in THPA-administrated apoE^-/- mice. Plasma analysis by enzymatic method or enzyme-linked immunosorbent assay (ELISA) showed that high-density lipoprotein-cholesterol (HDL-C) was decreased, whereas apolipoprotein A-I (apoA-I) levels were markedly increased by THPA. In addition, ELISA and spectrophotometric measurement showed that plasma levels of tumor necrosis factor-α, interleukin-1 and malondialdehyde were decreased in mice treated with THPA. Realtime polymerase chain reaction detection disclosed that the expression of several transporters involved in reverse cholesterol transport was induced by THPA, and the expression of hepatic ABCA1 and apoA-I, which play roles in the maturation of HDL-C, was also elevated in the THPA-treated groups. Moreover, THPA enhanced the expression of endothelial nitric oxide synthase (NOS), and reduced the expression of inducible NOS and lectin-like oxidized LDL receptor-1 in the aorta, suggesting that THPA can exert endothelial protection effects. In addition, the expression or activation of several proinflammatory factors in the aorta was suppressed by THPA. In conclusion, our results reveal the inhibitory effects of THPA on AS in apoE^-/- mice.

Evaluations of tumor growth rates and molecular biomarkers are traditionally used to assess new mouse models of human breast cancers. This study investigated the utility of diffusion weighted (DW)-magnetic resonance imaging (MRI) for evaluating cellular proliferation of new tumor models of triple-negative breast cancer, which may augment traditional analysis methods. Eleven human breast cancer cell lines were used to develop xenograft tumors in severe combined immunodeficient mice, with two of these cell lines exhibiting sufficient growth to be serially passaged. DW-MRI was performed to measure the distributions of the apparent diffusion coefficient (ADC) in these two tumor xenograft models, which showed a correlation with tumor growth rates and doubling times during each passage. The distributions of the ADC values were also correlated with expression of Ki67, a biomarker of cell proliferation, and hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor receptor-2 (VEGFR2), which are essential proteins involved in regulating aerobic glycolysis and angiogenesis that support tumor cell proliferation. Although phosphatase and tensin homolog (PTEN) levels were different between the two xenograft models, AKT levels did not differ nor did they correlate with tumor growth. This last result demonstrates the complexity of signaling protein pathways and the difficulty in interpreting the effects of protein expression on tumor cell proliferation. In contrast, DW-MRI may be a more direct assessment of tumor growth and cancer cell proliferation.

The ability to induce apoptosis is an important marker for cytotoxic antitumor agents. Some natural compounds have been shown to modulate apoptosis pathways that are frequently blocked in human cancers, and therefore, these compounds provide novel opportunities for cancer drug development. Phyllanthus, a plant genus of the family Euphorbiaceae, exhibits multiple pharmacological actions. Of these, Phyllanthus niruri extracts exhibit significant antitumor activity, which is consistent with the traditional medicinal use of this plant. To examine the apoptotic effects of a spray-dried extract of P. niruri (SDEPN), human hepatocellular carcinoma cells (HepG2, Huh-7), colorectal carcinoma cells (Ht29) and keratinocytes (HaCaT) were exposed to the extract for 4, 8 and 24 h. Flow cytometry and caspase-3 immunostaining were used to detect apoptosis, while analysis of variance was applied to identify significant differences between groups (P < 0.05). At all timepoints, the SDEPN induced significantly different cytotoxic effects for HepG2 and Huh-7 cells compared with control cells (P < 0.001). In contrast, the SDEPN had a protective effect on HaCaT cells compared with control cells at all timepoints (P < 0.001). In caspase-3 assays, activation was detected after cell death was induced in Huh-7 and HepG2 cancer cells by the SDEPN. In combination, these results indicate that the SDEPN is selectively toxic towards cancer cell lines, yet is protective towards normal cells.

An age-related decline in immune functions is referred to as immunosenescence. Mast cells play an important role in the immune system. However, it has not yet been determined if aging may affect mast-cell development. In the present study, we examined the age-related change in mast-cell development after myeloablation with 5-fluorouracil (5-FU) in senescence accelerated mice (SAMP1), which exhibit senescence-mimicking stromal cell impairment after 30 weeks of age. We found that aged mice with stromal cell impairment (30-36 weeks old) showed a lower recovery of the number of femoral mast-cell progenitors (colony-forming unit [CFU]-mast) (64% of steady state), whereas young mice (8-12 weeks old) showed a higher recovery (122% of steady state). Stromal cells influence mast-cell development by producing positive regulators such as stem cell factor (SCF) and negative regulators such as transforming growth factor-beta (TGF-β). The ratio of the gene expression of SCF to that of TGF-β (SCF/TGF-β βratio) indicates the balance of positive and negative regulation of mast-cell development. SCF/TGF-β βratio increased in both the young and aged mice after 5-FU treatment. However, the SCF/TGF-β βratio rapidly decreased in aged mice, whereas it remained high in young mice. The number of femoral CFU-mast in the S-phase after 5-FU treatment reflects the activation of positive-dominant regulation for mast-cell development by stromal cells. Aged mice showed lower recovery of the number of femoral CFU-mast in the S-phase (47% of steady state), whereas young mice showed a higher recovery (205% of steady state). These results suggest that mast-cell development declines with aging due to stromal-cell functional impairment, which contributes to immunosenescence.

The development of the prostate depends on a precise androgenic control, so sensible interferences may predispose this gland to develop prostatic diseases during life. These aspects are of interest and preoccupation, since human beings are exposed to a growing number of endocrine-disrupting chemicals with androgenic potential. Therefore, our aim was to evaluate the prostates of adult gerbils exposed to testosterone during intrauterine life. Serological, morphological, morphometric-stereologic, immunohistochemical and three-dimensional reconstruction analyses were used. We found that the testosterone effects were dose-dependent and more harmful to females, leading to the development of masculine characteristics, evidenced by an increased anogenital distance, and absence of vaginal opening and the ectopic development of prostatic tissue. Moreover, premalignant lesions, such as prostatic intraepithelial neoplasia, were observed in addition to inflammatory foci in the prostate. The results showed that the prenatal exposure to testosterone may affect the reproductive system, disrupting developmental processes and increasing susceptibility to the development of prostatic diseases in the Mongolian gerbil.

Dyslipidemia caused by ‘Western-diet pattern’ is a strong risk factor for the onset of diabetes. This study aimed to disclose the relationship between the serum metabolite changes induced by habitual intake of high-fat and high-cholesterol (HFHC) diet and the development of impaired glucose tolerance (IGT) and insulin resistance through animal models of Macaca mulatta. Sixteen M. mulatta (six months old) were fed a control diet or a HFHC diet for 18 months. The diet effect on serum metabolic profiles was investigated by longitudinal research. Islet function was assessed by intravenous glucose tolerance and hyperinsulinemic-euglycemic clamp test. Metabonomics were determined by ¹ H proton nuclear magnetic resonance spectroscopy. Prolonged diet-dependent hyperlipidemia facilitated visceral fat accumulation in liver and skeletal muscle and disorder of glucose homeostasis in juvenile monkeys. Glucose disappearance rate (K_Glu) and insulin response to the glucose challenge effects in HFHC monkeys were significantly lower than in control monkeys. Otherwise, serum trimethylamine-N-oxide (TMAO), lactate and leucine/isoleucine were significantly higher in HFHC monkeys. Sphingomyelin and choline were the most positively correlated with K_Glu (R² = 0.778), as well as negative correlation (R² = 0.64) with total cholesterol. The HFHC diet induced visceral fat, abnormal lipid metabolism and IGT prior to weight gain and body fat content increase in juvenile monkeys. We suggest that increased serum metabolites, such as TMAO, lactate, branched-chain amino acids and decreased sphingomyelin and choline, may serve as possible predictors for the evaluation of IGT and insulin resistance risks in the prediabetic state.

Corynebacterium pyruviciproducens is a newly discovered Corynebacterium species with no known pathogenic components such as diphtheria toxin and tuberculostearic acid, and it has similar biological properties to Propionibacterium acnes, but its role of immunoregulation is drawing people's attention. In this work, based on the role of macrophages in removal of pathogenic bacteria as a primary scavenger and particulate antigen-presenting cell, the stimulation of macrophages by C. pyruviciproducens was analyzed through detecting the levels of cytokine secretion and expression of membrane molecules, and the effect of C. pyruviciproducens in promoting antibody response to sheep red blood cells (SRBC) in vivo was detected. In vitro, C. pyruviciproducens led to a sharp release of interleukin-6 and tumour necrosis factor-α and encouraged the activation of macrophages including enhanced expressions of MHC-II, CD40, CD80 and CD86. In vivo, it enhanced the humoral immune response against SRBC, a particulate antigen. These observations suggest that C. pyruviciproducens, as an immunoregulator, can promote the host humoral immune response to pathogenic microorganisms by regulating macrophage function.

In the present work we studied synaptic protein concentrations in relation to behavioral performance. Long-Evans rats, aged 22-23 months, were classified for individual expression of place memory in the Morris water maze, in reference to young adults. Two main subgroups of aged rats were established: the Aged cognitively Unimpaired (AU) had search accuracy within the range (percent of time in training sector within mean+2 SEM) of young rats and the Aged cognitively Impaired (AI) rats had search accuracy below this range. Samples from the hippocampus and frontal cortex of all the AI, AU and young rats were analyzed for the expression of postsynaptic protein PSD-95 by Image J analysis of immunohistochemical data and by Western blots. PSD-95 expression was unchanged in the hippocampus, but, together with synaptophysin, was significantly increased in the frontal cortex of the AI rats. A significant correlation between individual accuracy (time spent in the training zone) and PSD-95 expression was observed in the aged group. No significant effect of age or PSD-95 expression was observed in the learning of a new position. All together, these data suggest that increased expression of PSD-95 in the frontal cortex of aged rats co-occurs with cognitive impairment that might be linked to functional alterations extending over frontal networks.

The aim of this study was to identify the roles and potential mechanisms of endoplasmic reticulum stress (ER stress), proapoptotic transcription factor C/EBP homologous protein (CHOP) and Bax in angiotensin II (Ang II)-induced cardiomyocyte apoptosis. Cultured neonatal rat cardiomyocytes were incubated with Ang II or antisense CHOP oligonucleotide which was used to inhibit CHOP expression. Expressions of ER chaperone immunoglobulin heavy chain-binding protein (BiP), CHOP and cytochrome c were examined by Western blotting. Mitochondrial membrane potential (MMP) was detected by a spectrofluorimeter. Apoptosis was analyzed with flow cytometry. Bax translocation was determined by double-labeling of immunofluorescence and Western blotting. Our results showed that Ang II-induced cardiomyocyte apoptosis was associated with the upregulations of BiP and CHOP, Bax translocation, MMP deplorization and cytochrome c release. These above effects were suppressed by antisense CHOP oligonucleotide. Furthermore, BiP and CHOP expressions, reactive oxygen species (ROS) production and cardiomyocyte apoptosis, which were upregulated by Ang II, were depressed by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin. From our results, ROS, ER stress and CHOP-mediated Bax translocation may be involved in Ang II-induced cardiomyocyte apoptosis.

Endometriosis, with a prevalence rate ranging from 6% to 10%, is the major contributor to pelvic pain and subfertility, and considerably reduces the quality of life in affected women. However, the pathogenesis of this disease remains largely unknown. The present study aimed to uncover the role of hyperperistalsis in the pathogenesis of endometriosis, by exploring the response of human endometrial stromal cells (ESCs) to the cyclic stretch in vitro. ESCs isolated from 18 different endometrium biopsies undergoing hysterectomy for myoma were subjected to uniaxial cyclic stretches with different magnitude and frequency using the Uniaxial Tension System. Expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) in stretched and unstretched ESCs were assessed by realtime quantitative polymerase chain reaction and Western blot. Production of prostaglandin E2 (PGE₂) in the culture medium was measured by enzyme-linked immunosorbent assay. The cyclic stretch mimicking hyperperistalsis in endometriosis (5% elongation at 4 cycles/min) stimulated quick up-regulations of COX-2 and mPGES-1 simultaneously on both transcriptional and translational levels, and delayed PGE₂ overproduction was also noted in ESCs. As the stretch magnitude or frequency increased, so did overexpression of COX-2 and PGE₂ (P < 0.05). By contrast, the cyclic stretch mimicking physiological peristalsis (3% elongation at 2 cycles/min) did not induce significant COX-2, mPGES-1 or PGE₂production within 12 h. Both COX-2 and mPEGS-1 are PGE₂ synthases, and the aberrant COX-2 and PGE₂ production play important roles in the pathogenesis of endometriosis. Therefore, the present findings revealed that increased stretch stimuli from the hyperperistalsis of endometriosis were capable of causing the aberrant COX-2 and PGE₂ expression in the endometrium by mechanotransduction, in a magnitude and frequency-dependent manner. It implied possible roles of hyperperistalsis in the pathogenesis of endometriosis, particularly in the view of COX-2 and PGE₂.

This study describes an innovative experimentally induced model of intervertebral disc degeneration. This innovative approach is based on the induction of extracellular matrix disorders in the intervertebral disc (IVD) using a diode laser. For this study, 15 one-year-old and five 30-month-old New Zealand White rabbits were used. Two procedures were tested to trigger IVD degeneration: needle aspiration (reference technique) and a laser approach. The IVD degeneration process was assessed 20, 40, 60, 90 and 120 days after surgery by X-ray radiography (IVD height), magnetic resonance imaging (MRI) (T2 intensity of IVD signal) and histological analysis using modified Boos’ scoring. Our data indicate that a marked IVD degeneration was found compared with sham-operated animals regardless of the procedure tested. A significant decrease in disc height on X-ray radiographs was first demonstrated. In addition, MRI disc signals were significantly reduced in both groups. Finally, a statistically significant increase in Boos’ scoring was found in both laser and aspiration-induced IVD degeneration. Interestingly, IVD degeneration induced by laser treatment was more progressive compared with aspiration. Moreover, the histological results indicated that laser-induced disc degeneration was quite similar to that obtained during the natural aging process as observed in 30-month-old rabbits. Our study describes the consistency of this innovative experimentally-induced animal model of IVD degeneration. The radiological, MRI and histological data confirm its relevance. The histological examination indicates that IVD degeneration induced by laser treatment is comparable to the degenerative process observed during the onset of spontaneous IVD degeneration. This model could be a useful tool to help us validate biomaterial-assisted, cell-based, regenerative medicine strategies for the prevention and treatment of IVD degeneration.