[Empirical analysis on lumbar disc herniation treated with "sinew-bone three needling technique" of Chinese medicine].

The paper presents professor WU Han-qing's experience in treatment of lumbar disc herniation (LDH) with "sinew-bone three needling technique" of Chinese medicine. Based on the theory of meridian sinew, the points are located by "three-pass method" in terms of the distribution of meridian sinew and syndrome/pattern differentiation. The cord-like muscles and adhesion are relieved by relaxing technique to work directly on the affected sites and alleviate the local compression to the nerve root. The needle technique is operated flexibly according to the affected regions involved, due to which, the needling sensation is increased while the safety ensured. As a result, the meridian qi is enhanced, the mind and qi circulation is regulated; and the clinical effect is improved.

Tributyltin impaired spermatogenesis and reproductive behavior in male zebrafish.

Tributyltin (TBT) was reported to affect sexual behavior and gametogenesis in fish. However, the modes of action involved are largely unclear. In order to elucidate the toxicological mechanisms of TBT in reproduction, zebrafish (Danio rerio) males were exposed to TBT at concentrations of 100 and 500 ng/L for 28 days. After exposure, the sperm count of the treated fish was sharply decreased though the testis weight and gonadosomatic index remained unchanged. Moreover, reduced number of spermatogonia and spermatozoa and increased spermatocytes were observed in TBT-treated fish by histological observation and PCNA-immunostaining. Increased number of apoptotic-positive spermatocytes was also present in TBT-treated fish, indicating an enhanced apoptosis in these cells. Consistent to decreased number of spermatogonia, down-regulated expressions of genes responsible for germ cell proliferation (cyclind1 and pcna) were observed in TBT-treated fish. In contrast, TBT elevated the expressions of genes involved in meiotic entry and maintenance (aldhla2, sycp3 and dmc1) while suppressed the mRNA level of gene responsible for terminus of meiotic entry (cyp26a1), in agreement with arrested meiosis and reduced sperm count. Furthermore, TBT significantly elevated the ratios of bax/bcl-2 and tnfrsf1a/tnfrsf1b in testis, which are markers for intrinsic- and extrinsic-apoptotic pathways, consistent with the enhanced TUNEL positive signals in spermatocytes. Moreover, TBT also significantly affected the parameter of reproductive behaviors in treated fish (reflected by decreased frequency of meeting, visits and time spent in spawning area). Consistently, the expressions of genes responsible for the modulation of reproductive behaviors in brain (such as cyp19a1b, kiss2, gnrh3 and ompb) were significantly down-regulated in treated-fish. Interestingly, disrupted reproductive behaviors of untreated female fish were also observed in the present study. The present study indicated that TBT might affect the reproduction of zebrafish male by disrupting the spermatogenesis and reproductive behavior of the fish.

Long-term exposure of xenoestrogens with environmental relevant concentrations disrupted spermatogenesis of zebrafish through altering sex hormone balance, stimulating germ cell proliferation, meiosis and enhancing apoptosis.

Environmental estrogens are capable of interfering with the spermatogenesis and fertility of fish. However in natural waters, these chemicals are more likely to occur as a combination rather than a single stressor. Whether and how the mixture of xenoestrogens with environmental relevant concentrations may affect fish spermatogenesis remains largely unknown. In this study, male zebrafish adults were administered to 17alpha-ethinylestradiol (EE2) and a mixture of xenoestrogens (Mix (E2, EE2, DES, 4-t-OP, 4-NP and BPA)), with the estrogenic potency equivalent to EE2. After a 60-day exposures, elevated mRNA levels of vitellogenin 1 (vtg1) and estrogen receptor 1 (esr1) in the liver of fish in both treated groups were observed. Moreover, the plasma level of E2 declined significantly in the Mix group and the ratio of 11-KT/E2 was significantly elevated in both treated groups. Consistently, the mRNA level of P450 side-chain cleavage (scc) in the EE2 group and ovarian type aromatase (cyp19a1a) in the Mix group was significantly suppressed. In addition, decreased gonadosomatic index and sperm count in the fish of Mix group were present. Furthermore, increased number of the proliferating germ cells (such as spermatogonia and spermatocytes) was observed in the fish of both groups, suggesting a stimulated germ cell proliferation and meiosis. Accordingly, both exposures significantly up-regulated the mRNA levels of genes in mitosis (cyclinb1) and meiosis (cyp26a1 in EE2 group, aldh1a2, cyp26a1, sycp3 and spo11 in Mix). In addition, decreased number of spermatozoa and increased number of TUNEL-positive signals were present in the testis of fish in the Mix group, indicating an enhanced apoptosis. Further analyses demonstrated the significant elevated expressions of tnfrsf1a and the ratio of tnfrsf1a/tnfrsf1b in the Mix group, suggesting an elevated apoptosis in the testis of fish in the Mix group via extrinsic pathway. The present study greatly extends our understanding of the underlying mechanisms of the reproductive toxicity of xenoestrogens on fish.

Tributyltin impaired reproductive success in female zebrafish through disrupting oogenesis, reproductive behaviors and serotonin synthesis.

Tributyltin (TBT), an organotin acting as aromatase (Cyp19a1) inhibitor, has been found to disrupt gametogenesis and reproductive behaviors in several fish species. However, few studies addressing the mechanisms underlying the impaired gametogenesis and reproduction have been reported. In this study, female adults of zebrafish (Danio rerio) were continuously exposed to two nominal concentrations of TBT (100 and 500 ng/L, actual concentrations: 90.8 ±â€¯1.3 ng/L and 470.3 ±â€¯2.7 ng/L, respectively) for 28 days. After exposures, TBT decreased the total egg number, reduced the hatchability and elevated the mortality of the larvae. Decreased gonadosomatic index (GSI) and altered percentages of follicles in different developmental stages (increased early-stage follicles and reduced mid/late-stage follicles) were also observed in the ovary of TBT-treated fish. TBT also lowered the plasma level of 17ß-estradiol and suppressed the expressions of cyp19a1a in the ovary. In treated fish, up-regulated expressions of aldhla2, sycp3 and dmc1 were present in the ovary, indicating an enhanced level of meiosis. The mRNA level of vtg1 was dramatically suppressed in the liver of TBT-treated fish, suggesting an insufficient synthesis of Vtg protein, consistent with the decreased percentage of mid/late-stage follicles in the ovaries. Moreover, TBT significantly suppressed the reproductive behaviors of the female fish (duration of both sexes simultaneously in spawning area, the frequency of meeting and the visit in spawning area) and down-regulated the mRNA levels of genes involved in the regulation of reproductive behaviors (cyp19a1b, gnrh-3 and kiss 2) in the brain. In addition, TBT significantly suppressed the expressions of serotonin-related genes, such as tph2 (encoding serotonin synthase), pet1 (marker of serotonin neuron) and kiss 1 (the modulator of serotonin synthesis), suggesting that TBT might disrupt the non-reproductive behaviors of zebrafish. The present study demonstrated that TBT may impair the reproductive success of zebrafish females probably through disrupting oogenesis, disturbing reproductive behaviors and altering serotonin synthesis. The present study greatly extends our understanding on the reproductive toxicity of TBT on fish.

Diethylstilbestrol arrested spermatogenesis and somatic growth in the juveniles of yellow catfish (Pelteobagrus fulvidraco), a fish with sexual dimorphic growth.

In fish, spermatogenesis and somatic growth are mainly regulated by hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-somatic (HPS) axes, respectively. Xenoestrogens have been reported to impair spermatogenesis in some fishes, and arrest somatic growth in some others, whereas, whether xenoestrogens are capable of disrupting spermatogenesis and somatic growth simultaneously in fish that exhibits sexual dimorphic growth is little known, and the underlying mechanisms remain poorly understood. In this study, male juveniles of yellow catfish (Pelteobagrus fulvidraco), which exhibits a sexual dimorphic growth that favors males, were exposed to diethylstilbestrol (DES) for 28 days. After exposure, DES significantly disrupted the spermatogenesis (decreased gonadal-somatic index (GSI) and germ cell number) and arrested the somatic growth (declined body weight) of the catfish juveniles. Gene expression and plasma steroid analyses demonstrated the suppressed mRNA levels of genes in HPG axis (gnrh-II, fshß, and lhß in the brain and dmrt1, sf1, fshr, cyp17a1, cyp19a1a, and cyp11b2 in the testis) and decreased 17ß-estrodial (E2) and 11-ketotestosterone (11-KT) levels in plasma. Further analysis revealed the arrested germ cell proliferation (cyclin d1), meiosis (dmc1, sycp3), and enhanced apoptosis (decreased bcl-2 and elevated bax/bcl-2 ratio) in the testis. Besides, DES also suppressed the mRNA levels of genes in HPS axis (ghrh, gh, and prl in the brain and ghr, igf1, igf2a, and igf2b in the liver). The suppressed HPG and HPS axes were thus supposed to disturb spermatogenesis and arrest somatic growth in yellow catfish. The present study greatly extended our understanding on the mechanisms underlying the toxicity of DES on spermatogenesis and somatic growth of fish.

Exposure to mercuric chloride induces developmental damage, oxidative stress and immunotoxicity in zebrafish embryos-larvae.

Mercury (Hg) is a widespread environmental pollutant that can produce severe negative effects on fish even at very low concentrations. However, the mechanisms underlying inorganic Hg-induced oxidative stress and immunotoxicity in the early development stage of fish still need to be clarified. In the present study, zebrafish (Danio rerio) embryos were exposed to different concentrations of Hg2+ (0, 1, 4 and 16µg/L; added as mercuric chloride, HgCl2) from 2h post-fertilization (hpf) to 168hpf. Developmental parameters and total Hg accumulation were monitored during the exposure period, and antioxidant status and the mRNA expression of genes related to the innate immune system were examined at 168hpf. The results showed that increasing Hg2+ concentration and time significantly increased total Hg accumulation in zebrafish embryos-larvae. Exposure to 16µg/L Hg2+ caused developmental damage, including increased mortality and malformation, decreased body length, and delayed hatching period. Meanwhile, HgCl2 exposure (especially in the 16µg/L Hg2+ group) induced oxidative stress affecting antioxidant enzyme (CAT, GST and GPX) activities, endogenous GSH and MDA contents, as well as the mRNA levels of genes (cat1, sod1, gstr, gpx1a, nrf2, keap1, hsp70 and mt) encoding antioxidant proteins. Moreover, the transcription levels of several representative genes (il-1ß, il-8, il-10, tnfα2, lyz and c3) involved in innate immunity were up-regulated by HgCl2 exposure, suggesting that inorganic Hg had the potential to induce immunotoxicity. Taken together, the present study provides evidence that waterborne HgCl2 exposure can induce developmental impairment, oxidative stress and immunotoxicity in the early development stage of fish, which brings insights into the toxicity mechanisms of inorganic Hg in fish.

Reproductive toxicity of inorganic mercury exposure in adult zebrafish: Histological damage, oxidative stress, and alterations of sex hormone and gene expression in the hypothalamic-pituitary-gonadal axis.

Mercury (Hg) is a prominent environmental contaminant that causes a variety of adverse effects on aquatic organisms. However, the mechanisms underlying inorganic Hg-induced reproductive impairment in fish remains largely unknown. In this study, adult zebrafish were exposed to 0 (control), 15 and 30µg Hg/l (added as mercuric chloride, HgCl2) for 30days, and the effects on histological structure, antioxidant status and sex hormone levels in the ovary and testis, as well as the mRNA expression of genes involved in the hypothalamic-pituitary-gonadal (HPG) axis were analyzed. Exposure to Hg caused pathological lesions in zebrafish gonads, and changed the activities and mRNA levels of antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx)) as well as the content of glutathione (GSH) and malondialdehyde (MDA). In females, although ovarian 17ß-estradiol (E2) content remained relatively stable, significant down-regulation of lhß, gnrh2, gnrh3, lhr and erα were observed. In males, testosterone (T) levels in the testis significantly decreased after Hg exposure, accompanied by down-regulated expression of gnrh2, gnrh3, fshß and lhß in the brain as well as fshr, lhr, ar, cyp17 and cyp11b in the testis. Thus, our study indicated that waterborne inorganic Hg exposure caused histological damage and oxidative stress in the gonads of zebrafish, and altered sex hormone levels by disrupting the transcription of related HPG-axis genes, which could subsequently impair the reproduction of fish. Different response of the antioxidant defense system, sex hormone and HPG-axis genes between females and males exposed to inorganic Hg indicated the gender-specific regulatory effect by Hg. To our knowledge, this is the first time to explore the effects and mechanisms of inorganic Hg exposure on reproduction at the histological, enzymatic and molecular levels, which will greatly extend our understanding on the mechanisms underlying of reproductive toxicity of inorganic Hg in fish.

Effects of recombinant human leptin administration on hepatic lipid metabolism in yellow catfish Pelteobagrus fulvidraco: in vivo and in vitro studies.

The present study was conducted to investigate the effects and mechanism of leptin influencing lipid metabolism in yellow catfish Pelteobagrus fulvidraco. To this end, hepatic lipid (in vivo experiment) and intracellular triglyceride (TG) (in vitro experiment) content, the activities and/or expression level of several enzymes (CPT-1, 6PGD, G6PD, FAS, ME and ICDH) as well as the mRNA expression of transcription factors (PPARα, PPARγ and SREBP-1) involved in lipid metabolism were determined. Using the primary hepatocytes of yellow catfish, specific inhibitors AG490 (JAK-STAT inhibitor) and wortmannin (IRS-PI3K inhibitor) were used to explore the signaling pathways of leptin effects on lipid metabolism. Intraperitoneal injection of recombinant human leptin (rt-hLEP) significantly reduced hepatic lipid content, activities of lipogenic enzymes (6PGD, G6PD, ME, ICDH and FAS) as well as mRNA levels of 6PGD, G6PD, FAS, PPARγ and SREBP-1 genes, but up-regulated activity and mRNA level of CPT-1 and PPARα. Using primary hepatocytes, rt-hLEP incubation also reduced intracellular TG content, mRNA levels of G6PD and PPARγ genes, but enhanced mRNA levels of PPARα, CPT-1 and SREBP-1. Leptin-induced effects could partially be reversed by specific inhibitors AG490, suggesting that JAK-STAT signaling pathways played important roles in the process of leptin-induced changes in lipid metabolism. Wortmannin significantly suppressed the decrease of TG content induced by leptin, reflecting that IRS-PI3K was involved in the leptin-mediate changes as well. To our knowledge, the present study provides, for the first time, evidence that rt-hLEP can increase lipolysis and reduce lipogenesis at the both enzymatic and molecular levels in fish with the combination of in vivo with in vitro studies, which serves to increase our understanding into the roles and mechanisms of leptin regulating lipid metabolism in fish.

Effects of copper and cadmium on lipogenic metabolism and metal element composition in the javelin goby (Synechogobius hasta) after single and combined exposure.

The present study was performed to determine the effects of single and combined exposure of copper (Cu) and cadmium (Cd) on lipogenic metabolism and metal element composition of javelin goby Synechogobius hasta. Two hundred and forty uniform-sized S. hasta (initial mean weight 20.3 ± 0.3 g [mean ± SEM throughout]; initial body length 15.2 ± 0.2 cm) were randomly assigned to 12 fiberglass tanks (water volume 300 l) with 20 fish/tank. The fish were exposed to four treatments with different Cu and Cd concentration for 30 days, respectively: (1) control (without extra Cu and Cd addition), (2) Cu (nominal concentrations of 77 µg/l), (3) Cd (79 µg/l), and (4) Cu + Cd (Cu/Cd coexposure). Growth decreased, but hepatosomatic index, viscerosomatic index, and lipid content increased after metal exposure. Staining with Oil Red O and haematoxylin and eosin showed extensive alterations in liver of metals-exposed fish. Metal exposure influenced the accumulation of metal elements (Cu, Cd, iron, zinc, and manganese) in several tissues (muscle, gill, intestine, liver, and spleen) and increased hepatic 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, malic enzyme, isocitrate dehydrogenase, and fatty acid synthase activities. The results of the present study indicated that the changes in lipogenic metabolism and metal element compositions of fish under Cu and Cd coexposure could not be explained by synergism of the addition of the effects observed in singly Cu- or Cd-exposed fish. To our knowledge the present study, for the first time, investigated the effects of Cu and Cd coexposure on hepatic lipogenic metabolism and metal element compositions in a wide range of tissues and organs in fish, which provided new evidence for Cu and Cd interactions in fish.

Molecular cloning and mRNA tissue expression of thyroid hormone receptors in yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta.

Thyroid hormones (THs) play a pivotal role in many physiological functions in vertebrates, including fish. Their effects are mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. In this study, full-length cDNA sequences of TRs from yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta were cloned and their mRNA tissue expression profiles were determined. In P. fulvidraco, the validated cDNAs encoding for TRα and TRß were 1789 and 1848 bp in length, encoding peptides of 401 and 378 amino acid residues, respectively. In addition, a TRß spliced variant (named P. fulvidraco-TRßv), containing a 60-bp insertion, was detected. In S. hasta, cDNAs encoding for TRαA, TRαB and TRß were 1827, 2295 and 2258 bp in length, encoding peptides of 401, 409 and 393 amino acid residues, respectively. The phylogenetic analysis revealed that TRα and TRß cDNAs grouped into two separate clusters with other vertebrate counterparts and two TRα sequences grouped separately, suggesting that the two TRαs derived from paralogous genes that might arise during a teleost-specific genome duplication event. All TR mRNAs were detected in various tissues sampled. The mRNA levels of both TRα and TRß from P. fulvidraco were the highest in brain, followed by liver, and lowest in heart, intestine, muscle, gill and spleen. However, in S. hasta, TRαA, TRαB and TRß showed the highest mRNA levels in brain and lowest in muscle. Identification and mRNA tissue expression of TR genes from P. fulvidraco and S. hasta provide an initial step towards understanding their biological roles in the two fish species.

Differential effect of waterborne cadmium exposure on lipid metabolism in liver and muscle of yellow catfish Pelteobagrus fulvidraco.

The present study was conducted to investigate the effect of waterborne cadmium (Cd) exposure on lipid metabolism in liver and muscle of juvenile yellow catfish Pelteobagrus fulvidraco. Yellow catfish were exposed to 0 (control), 0.49 and 0.95 mg Cd/l, respectively, for 6 weeks, the lipid deposition, Cd accumulation, the activities and expression level of several enzymes as well as the mRNA expression of transcription factors involved in lipid metabolism in liver and muscle were determined. Waterborne Cd exposure reduced growth performance, but increased Cd accumulation in liver and muscle. In liver, lipid content, the activities and the mRNA expression of lipogenic enzymes (6-phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), fatty acid synthetase (FAS)) and lipoprotein lipase (LPL) activity increased with increasing waterborne Cd concentrations. However, the mRNA expressions of LPL and peroxisome proliferators-activated receptor (PPAR) α were down-regulated by Cd exposure. Carnitine palmitoyltransferase 1 (CPT1) activity as well as the mRNA expressions of CPT1 and PPARγ showed no significant differences among the treatments. In muscle, lipid contents showed no significant differences among the treatments. The mRNA expression of 6PGD, FAS, CPT1, LPL, PPARα and PPARγ were down-regulated by Cd exposure. Thus, our study indicated that Cd triggered hepatic lipid accumulation through the improvement of lipogenesis, and that lipid homeostasis in muscle was probably conducted by the down-regulation of both lipogenesis and lipolysis. Different variation patterns of lipid metabolism to waterborne Cd exposure indicated the tissue-specific regulatory effect of lipid metabolism under waterborne Cd exposure. To our knowledge, the present study provides, for the first time, evidence that waterborne chronic Cd exposure can disturb the normal processes of lipid metabolism at both the enzymatic and molecular levels, and in two tissues (the liver and muscle).

Protective effects of calcium pre-exposure against waterborne cadmium toxicity in Synechogobius hasta.

The present study was performed to evaluate the effects of calcium (Ca) pre-exposure and then waterborne cadmium (Cd) exposure on metal element accumulation, enzymatic activities, histology, and ultrastructure in Synechogobius hasta and test the hypothesis that Ca could protect against Cd-induced toxicity in the fish species. Three hundred sixty fish [initial mean weight 25.5 ± 0.1 g (mean ± SEM)] were stocked in 18 circular fiberglass tanks (water volume: 300 l), 9 of which were pre-exposed to Ca at a rate of 400 mg Ca/l for 9 days and then exposed to concentrations of 0, 79.3, and 158.6 µg Cd/l for 9 days. Another 9 tanks were cultured in natural seawater (no extra Ca addition) for 9 days and then exposed to concentrations of 0, 79.3, and 158.6 µg Cd/l for 9 days. Both Ca pre-exposure and then waterborne Cd exposure influenced the accumulation of metal elements [cadmium (Cd), copper, zinc, and iron] in several tissues (muscle, gill, liver, spleen, and intestine), changed hepatic intermediary metabolism, and induced histological and ultrastructural alterations in tissues. In general, Ca pre-exposure seemed to mitigate the severity of Cd-induced mortality and histopathological injuries indicating that Ca pre-exposure had the capacity to decrease Cd toxicity in S. hasta.