Otoprotective effects of erythropoietin on Cdh23erl/erl mice.

The Cdh23(erl/erl) mice are a novel mouse model for DFNB12 and are characterized by progressive hearing loss. In this study, erythropoietin (EPO) was given to the Cdh23(erl/erl) mice by intraperitoneal injection every other day from P7 for 7 weeks. Phosphate-buffered saline-treated or untreated Cdh23(erl/erl) mice were used as controls. Auditory-evoked brainstem response (ABR) thresholds and distortion product oto-acoustic emission (DPOAE) were measured in the mouse groups at the age of 4, 6 and 8 weeks. The results show that EPO can significantly decrease the ABR thresholds in the Cdh23(erl/erl) mice as compared with those of the untreated mice at stimulus frequencies of click, 8-, 16- and 32-kHz at three time points. Meanwhile, DPOAE amplitudes in the EPO-treated Cdh23(erl/erl) mouse group were significantly higher than those of the untreated groups at f2 frequency of 15383 Hz at the three time points. Furthermore, the mean percentage of outer hair cell loss at middle through basal turns of cochleae was significantly lower in EPO-treated Cdh23(erl/erl) mice than in the untreated mice (P<0.05). This is the first report that EPO acts as an otoprotectant in a DFNB12 mouse model with progressive hearing loss.

A new mouse mutant of the Cdh23 gene with early-onset hearing loss facilitates evaluation of otoprotection drugs.

We report a novel mutation (erlong, erl) of the cadherin 23 (Cdh23) gene in a mouse model for DFNB12 characterized by progressive hearing loss beginning from postnatal day 27 (P27). Genetic and sequencing analysis revealed a 208 T >C transition causing an amino-acid substitution (70S-P). Caspase expression was upregulated in mutant inner ears. Hearing was preserved (up to 35-dB improvement) in pan-caspase inhibitor Z-VAD-FMK-treated mutants compared with untreated mutants (P<0.05). Outer hair cell (OHC) loss in the cochleae of Z-VAD-FMK-treated mutants was significantly reduced compared with those of untreated mice. Thus, the erl mutation can lead to hearing loss through apoptosis. This is the first genetic mouse model of hearing loss shown to respond to otoprotective drug therapy. The short interval from initial hearing loss to deafness (P27-P90) makes this model ideal for screening and validating otoprotective drugs.

Esculentoside A inhibits tumor necrosis factor, interleukin-1, and interleukin-6 production induced by lipopolysaccharide in mice.

Esculentoside A, a kind of saponin isolated from the root of the Chinese herb Phytolaca esculenta, is reported to possess potent anti-inflammatory effects in acute and chronic experimental models. In the present study, we investigated the effects of esculentoside A on the production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) induced by lipopolysaccharide (LPS) in mice. In vitro experiments demonstrated that esculentoside A (0.1-10 mumol/l) significantly reduced the release of TNF from the peritoneal macrophages derived from mice pretreated with thioglycolate. IL-1 and IL-6 secretion was also obviously inhibited in a concentration-dependent manner by esculentoside A from 0.01 to 10 mumol/l. In vivo experiments demonstrated that detectable TNF was observed 0.25 h after injection, was maximal at 0.5 h, and returned to baseline at 4 h. Maximal production of IL-1 and IL-6 were observed to be 1 and 2 h, respectively, after injection of LPS. Pretreatment of mice with 5, 10, or 20 mg/kg esculentoside A once a day for 7 consecutive days dose-dependently decreased the TNF, IL-1 and IL-6 levels in the sera of mice following LPS challenge. TNF, IL-1, and IL-6 are important cytokines involved in the pathogenesis of inflammatory lesions. Inhibition of inflammatory cytokine production may contribute to the anti-inflammatory effects of esculentoside A.

Effects of Phytolacca acinosa polysaccharides I with different schedules on its antitumor efficiency in tumor bearing mice and production of IL-1, IL-2, IL-6, TNF, CSF activity in normal mice.

Effects of Phytolacca acinosa polysaccharides I (PAP-I), 5 approximately 40 mg/kg in timing of 7 times/wk, 3 times/wk and 1 time/wk on their antitumor efficiency in Sarcoma-180 bearing mice were comparatively investigated. The results confirmed that PAP-I (10 mg/kg, 3 times/wk) reached its optimal antitumor efficiency. Concanavalin A-, lipopolysaccharides-induced lymphocyte proliferation and the IL-2 production were tested in normal mice which were treated with PAP-I, 5 approximately 50 mg/kg in timing of 1 time/wk and 3 times/wk. The results showed that PAP-I could augment lymphocyte proliferation and IL-2 production in the group treated with PAP-I in timing of once a week. However, in the group 3 times/wk, PAP-I could significantly weaken lymphocyte proliferation and IL-2 production. Further studies on IL-1, TNF and IL-6 secreted from macrophages and the level of CSF activity in serum of normal mice with different schedules showed that PAP-I (10 mg/kg, 3 time/wk) was the best one in regulating the production of IL-1, TNF, IL-6 and CSF activity. M-CSF was confirmed in the serum by using monoclonal antibody of IL-3, GM-CSF and polyclonal antibody of M-CSF. These results suggested that the antitumor effect of PAP-I, may be mainly related to its augmenting effect on macrophages in mice.

Brevitaxin, a new diterpenolignan from the bark of Taxus brevifolia.

The first terpenolignan, brevitaxin [1], has been isolated from the bark of Taxus brevifolia. Identification was carried out using spectral methods, and the regiochemistry and cytotoxicity of 1 are discussed.

Effects of Phytolacca acinosa polysaccharides I combined with interleukin-2 on the cytotoxicity of murine splenocytes against tumor cells.

Phytolacca acinosa polysaccharides I (PAP-I), a kind of purified polysaccharides, isolated from Phytolacca acinosa Roxb was found to significantly augment the cytotoxicity of murine splenocytes and interleukin-2 (IL-2) activated splenocytes against P815 tumor cells in vitro. The optimal concentration of PAP-I was 1 microgram.ml-1 and the peak level of the cytotoxicity against P815 tumor cells was reached on d 3-5. The supernatants collected from splenocytes cultured with PAP-I alone or in combination with IL-2 showed no effect on the cytotoxicity against P815 tumor cells. Splenocytes from mice injected ip with PAP-I, 5, 10 and 50 mg.kg-1, thrice a week produced more cytotoxicity against P815 and L929 tumor cells compared with the control group. PAP-I ip was shown to significantly increase IL-2 activated killer cell activity (LAK) against P815 tumor cells. The higher the dosage of PAP-I, the more potent the LAK activity was observed. These results confirmed that PAP-I can augment the cytotoxicity of murine splenocyte against tumor cells and LAK activity and warranted further evaluation of its clinical usefulness.

[Effect of Phytolacca acinosa polysaccharides I on production of colony-stimulating factors of mouse splenocytes in vitro].

Phytolacca acinose polysaccharides I (PAP-I), M(r) = 10,000, activated mouse splenocytes to produce colony-stimulating factors (CSF) in vitro. The level of CSF was tested by [3H]TdR uptaken by bone marrow cells and rmGM-CSF was used as standard. PAP-I (10-500 micrograms.ml-1) increased CSF production of the splenocytes treated with or without concanavalin A. When the concentration of PAP-I was 100 micrograms.ml-1, the level of CSF was about equivalent to that of rmGM-CSF 11.8 +/- 1.8 ng.ml-1. After a 3-d incubation of PAP-I with the splenocytes, CSF was assayed. The longer the incubation, the higher were the CSF concentrations. The CSF type in supernants of splenocytes induced by PAP-I was determined by IL-3 McAb, GM-CSF McAb, and M-CSF PcAb. The type of CSF was found to be interleukin-3.

Effects of Phytolacca acinosa polysaccharides I on immune function in mice.

Radioactivities of [3H]TdR uptaken by splenocytes and released from [3H]TdR-labeled YAC-1 cell line were measured to determine the degree of lymphocyte proliferation and natural killer (NK) cell activity. Seven days after mice treated with Phytolacca acinosa polysaccharides I (PAP-I) 5-50 mg.kg-1, the NK cell activity, and lymphocyte proliferation induced by Con A 5 micrograms.ml-1 or lipopolysaccharides 10 micrograms.ml-1 were significantly augmented. Splenocytes from mice treated with ip PAP-I 5-50 mg.kg-1 were incubated with Con A 5 micrograms.ml-1 for 24 h to induce interleukin-2 (IL-2) and for 40 h to induce NK cytotoxic factor (NKCF). Radioactivities of [3H]TdR uptaken by CTLL-2 cell line and YAC-1 cell line were used to measure the IL-2 and NKCF activities, respectively. PAP-I enhanced the production of IL-2 and NKCF. These results suggest that PAP-I augments the immunological functions in vivo.

[Descending cord dorsum potentials evoked by stimulation at "Yong Quan" acupoint].

Segmental spinal cord potential (Y-sCDP) evoked by stimulation at "Yong Quan" acupoint of Sprague-Dawley rat vanished gradually when the recording electrode was moved rostrally along the dorsal surface of the spinal cord. When the recording electrode was placed more rostrally, for example at C6, another positive slow potential, Y-dCDP, could be recorded. Y-dCDP disappeared completely when the spinal cord had been sectioned at a higher cervical level. Electrolytic lesions of ventral periaqueductal gray (PAG) could also decrease remarkably the amplitude of Y-dCDP. This facet suggests that the PAG, an analgesia related nucleus, is involved in the generation of Y-dCDP induced by stimulation at "Yong Quan" acupoint.

[Effects of Phytolacca acinosa polysaccharides II on lymphocyte proliferation and colony stimulating factor production from mice splenocytes in vitro].

Phytolacca acinosa polysaccharides II (PAP-II), a kind of polysaccharides isolated from Phytolacca acinosa Roxb with MW 40 kDa, on lymphocyte proliferation and colony stimulating factor (CSF) production from splenocytes in vitro, was studied. The radioactivities of [3H] TdR uptake by lymphocyte and bone marrow cells were used to determine the ability of lymphocyte proliferation and CSF production respectively. PAP-II was found to significantly augment splenocyte proliferation in a dose-dependent fashion, and significantly enhance Con A (1, 2.8 micrograms.ml-1) and LPS (3, 10, 30 micrograms.ml-1) induced lymphocyte proliferation at concentration of 31-125 micrograms.ml-1. As the concentration of PAP-II increased, significant suppression of Con A-induced lymphocyte proliferation was observed. Induction of CSF by PAP-II from splenocyte was confirmed at the present study. The optimal dosage was 100 micrograms.ml-1 and the optimal effect occurred on day 5. These results suggest that PAP-II can augment immunological function and enhance hematopoiesis.

[Effects of Phytolacca acinosa polysaccharide on splenic lymphocyte proliferation and cytokine secretion from splenic lymphocyte and macrophage].

The effects of Phytolacca acinosa polysaccharides I (PAP-I), a polysaccharide extracted from Phytolacca acinosa Roxb: on splenic lymphocyte proliferation and cytokines production from splenic lymphocyte and macrophage were studied. Lipopolysaccharides (LPS) and PAP-I were found to significantly augment splenic lymphocyte proliferation of normal BALB/c, nude BALB/c and NC mice in vitro, but concanavalin A (Con A) was shown to stimulate only normal BALB/c and nude BALB/c splenic lymphocyte proliferation. Also, PAP-I significantly enhanced Con A or LPS-induced lymphocyte proliferation and mixed lymphocyte reaction. Significant enhancement of colony stimulating factor (CSF) production was observed from splenic lymphocyte of normal BALB/c and nude BALB/c mice but not from NC mice when treated with PAP-I for 5 d. PAP-I was shown to significantly enhance interleukin-2 (IL-2) production from normal mice splenocyte and Con A stimulated normal mice splenocyte in a concentration-dependent fashion. Supernatant of PAP-treated macrophage (M phi) were collected and CSF activity was tested. The results confirmed that PAP-I can significantly stimulate M phi to secret CSF activity on d 1. The supernatant also contained a cytokine which exhibited a synergistic action with recombinant murine granular-macrophage CSF (RMGM-CSF) to stimulate mice bone marrow cell proliferation. PAP-I, 5-50 mg.kg-1, ip can enhanced splenic lymphocyte proliferation and IL-2 production. These findings indicate that PAP-I can augment immunologic function in vitro and in vivo.

[Inhibitory effect of esculentoside A on platelet activating factor released from calcimycin induced rat peritoneal macrophages].

Platelet activating factor (PAF) is a kind of inflammatory mediator. We used the method of aggregation of washed rabbit platelet to study the effect of esculentoside A (EsA) on the release of PAF from calcimycin (A23187)stimulated rat peritoneal macrophages and found that the release of PAF was inhibited by EsA in a time and dose dependent manner. The IC50 of EsA for the inhibition of PAF release was 1.5 mumol/L. Under the same condition, the release of PAF was also inhibited by mepacrine at 100 mumol/L. The results indicate that EsA is a potent inhibitor of PAF synthesis in rat peritoneal macrophages.

[Effects of Phytolacca acinosa polysaccharides I on cytotoxicity of macrophages and its production of tumor necrosis factor and interleukin 1].

The in vivo effects of Phytolacca acinosa polysaccharides I (PEP-I) on immunologic cytotoxicity of mouse peritoneal macrophages and its production of tumor necrosis factor (TNF) and interleukin 1 (IL-1) were studied. PEP-I 80, 160 mg/kg was given ip twice every 4 d. Both doses were found to have significant enhancing activity on macrophages cytotoxicity against S180 sarcoma cells and malignant transformed fibroblast L929 cells. Peritoneal activated macrophages were incubated with LPS for 2 and 24 h to induce TNF and IL-1, respectively. The TNF and IL-1 activities were tested from cytotoxicity against L929 cells in an absorbance assay of enzymatic reaction and proliferation of thymocytes co-stimulated assay separately. The optimal time for TNF production was found on d 8. Significant increases in TNF and IL-1 were observed. In comparison of the effect of PEP-I on TNF with that of known priming agent BCG, there was no difference between these two, but PEP-I had a high effect on IL-1. These results suggest that cytotoxicity of macrophages primed by PEP-I is closely related to its TNF and IL-1 production.

[Immunosuppressive and antiinflammatory activities of tripterine].

Tripterine is one of the active components isolated from Tripterygium wilfordii Hook.f. In this study tripterine was shown to inhibit the antibody response of mouse splenocytes to SRBC both in vitro and in vivo either by primary or secondary stimulation. Tripterine was also found to significantly inhibit cotton pellet induced granuloma growth in rats and depress the delayed hypersensitive reaction of mouse skin to dinitrochlorobenzene (DNCB). Besides, it also increased the sleeping time of mouse induced by pentobarbital sodium. From the results stated above, it appears that tripterine might be useful in the treatment of inflammatory diseases or autoimmune diseases.