ABSTRACT
AIM: To observe the effects of berberine (Ber) on enterocyte apoptosis in septic mice and its pos-sible mechanism.METHODS: Male C57BL/6 mice (8 ~10 weeks old) were randomly divided into sham group, cecal ligation and puncture (CLP) group, CLP +Ber group and sham +Ber group.The mice in CLP group underwent CLP ope-ration, and the mice in sham groups suffered a similar operation except the ligation and puncture.After the sham or CLP operation, the mice were administered intragastrically with distilled water or berberine (50 mg/kg) within 2 h.After 20 h, the mice were killed with excess pentobarbital sodium and the ileum tissues were removed.The histological changes of the intestine were observed and the enterocyte apoptosis was examined by determining the protein level of cleaved caspase-3. Furthermore, mitochondrial Bax, cytoplasm cytochrome C (Cyt C) and the total proteins of Bcl-2, Fas, FasL and Fas-as-sociated protein with death domain (FADD) were examined by Western blot.The mRNA expression of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) was measured by real-time PCR.RESULTS: The extensive ileum injuries, including remarkably increased leukocytes and necrosis of intestinal villus were observed 20 h after CLP.In CLP group, the protein levels of cleaved caspase-3, cytoplasm Cyt C, as well as Fas, FasL were significantly increased, but the Bcl-2 level was decreased.Bax translocation into mitochondria was promoted.However, FADD was not changed significantly.The mRNA expression of TH and DBH was also increased sharply in CLP group.On the contrary, treatment with berberine made a considerable alleviating alteration in the ileum of the septic mice.CONCLUSION: Treatment with berberine pro-vides protective effects on intestinal injury in septic mice by reducing enterocyte apoptosis, and its possible mechanism may be involved in the inhibition of the endogenous and exogenous apoptosis pathways.
ABSTRACT
AIM:To observe the effect of B-HT933, a selective α2-adrenoceptor agonist, on lipopolysaccha-ride ( LPS )-induced TNF-αproduction in neonatal rat cardiomyocytes and to explore the underlying mechanisms . METHODS:The neonatal rat cardiomyocytes were cultured .The localization of α2A-adrenoceptor in the cardiomyocytes was examined by immunofluorescence staining .The cardiomyocytes were exposed to LPS or/and B-HT933 for different time.The level of TNF-αin the supernatants and the mRNA expression of TNF-αwere detected by ELISA and real-time PCR, respectively.In addition, LPS-associated signal molecules in the cardiomyocytes were also examined by Western blotting.RESULTS: Immunofluorescence staining showed that α2A-adrenoceptors were localized in the cardiomyocytes . LPS stimulated TNF-αproduction in the cardiomyocytes in a dose and time-dependent manner .B-HT933 pretreatment sig-nificantly inhibited the expression of TNF-αat mRNA and protein levels in LPS-treated cardiomyocytes .Furthermore, LPS exposure induced IκBαand p38 phosphorylation in cardiomyocytes and only IκBαphosphorylation was prevented by B-HT933 treatment.CONCLUSION:α2A-adrenoceptors are present in neonatal rat cardiomyocytes and its agonist B -HT933 inhibits LPS-induced TNF-αproduction in cardiomyocytes via suppressing IκBαphosphorylation .
ABSTRACT
[ ABSTRACT] AIM:To observe the effects of berberine and yohimbine on splenocyte apoptosis in septic mice and underlying mechanisms.METHODS:The mice were subjected to cecal ligature and puncture ( CLP) .The drugs or vehi-cle were given intragastrically 2 h after the surgery according to the following 5 groups:sham, CLP, CLP+berberine, CLP+yohimbine, and CLP+berberine+yohimbine.The apoptosis of splenocytes stained by TUNEL was observed under laser scanning confocal microscope 20 h after CLP.The splenic lymphocytes were isolated and observed using flow cytometry. The activities of caspase-3, caspase-8 and caspase-9 in splenic lymphocytes were detected, and the expression of Fas, Bim, Bcl-2 and Bax in the splenocytes was also determined by Western blotting.RESULTS:The TUNEL staining showed that the apoptotic rate of the splenocytes in septic mice 20 h after CLP was significantly higher than that in sham and CLP+yohimbine groups (P<0.05).Compared with CLP group, the proportion of apoptotic cells was decreased in septic mice in CLP+berberine+yohimbine and CLP+yohimbine groups ( P<0.05) .Flow cytometry analysis demonstrated the similar results in the apoptosis of splenocytes and T lymphocytes.However, only yohimbine treatment reduced the apoptosis of B lymphocytes in the spleen of sepsis-challenged mice.Compared with CLP group, caspase-9 activity was significantly re-duced in CLP+berberine group (P<0.05), the activities of caspase-3, caspase-8 and caspase-9 were all statistically re-duced (P<0.05) in CLP+yohimbine group and CLP+yohimbine+berberine group.CLP significantly increased the ex-pression of cytosolic Fas, Bim and mitochondrial Bax in the splenocytes, and decreased Bcl-2 expression compared with sham group.Compared with CLP group, the expression of cytosolic Bim and mitochondrial Bax in CLP+berberine group were reduced (P<0.05).Fas expression decreased only in CLP+yohimbine group (P<0.05).Berberine combined with yohimbine reduced the expression of cytosolic Fas, Bim and mitochondrial Bax in the septic mouse splenocytes ( P <0.05).CONCLUSION:Yohimbine reduces sepsis-induced splenic lymphocyte apoptosis in mice by inhibiting Fas expres-sion and in turn blocking both extrinsic and intrinsic apoptosis pathways.Berberine reduces Bim expression and inhibits caspase-9 activation, but not caspase-3 activation and apoptosis in the septic mouse splenocytes.Berberine combined with yohimbine reduces splenocyte apoptosis in the septic mice by inhibiting both extrinsic and intrinsic apoptotic pathways.