The effect of secondary cholestasis on the CD68-positive and CD163-positive macrophage population, cellular proliferation, and apoptosis in rat testis.

The pathogenesis of hypogonadism in male patients with cirrhosis is complex and not well explained. Systemic infection and inflammation can inhibit testicular functions of endocrine and spermatogenesis. The acute inflammation induced by lipopolysaccharide can cause testicular acute inflammation. Both clinical and animal experimental data indicate that the developing process of cholestasis/cirrhosis can lead to endotoxemia. Little is known about the long-term effects of cholestasis on the intratesticular macrophage population, cellular proliferation and apoptosis. A rat model of secondary cholestasis caused by common bile duct ligation (CBDL) was used to evaluate the impact of cholestasis on them, and the influence of biliary decompression (choledochoduodenostomy). Endotoxemia occurred in animals at 20 days CBDL (20dCBDL) and 30 days CBDL (30dCBDL), but disappeared after 30 days biliary decompression in rats with CBDL. There was a considerable increase in the numbers of intratesticular CD68(+) and CD163(+) macrophages following CBDL. After biliary decompression, CD68(+) macrophage numbers decreased, but remained higher than that of controls; meanwhile, CD163(+) remained elevated only in rats with 30dCBDL. After CBDL, there was a progressive decrease in the expression of Bcl-2 protein and in proliferating cell nuclear antigen (PCNA(+)) cells, and a dramatic increase in the expression of Bax, active caspase-3 and apoptotic cells. These data suggest that secondary cholestasis expands the population of CD68(+) and CD163(+) macrophages in the testicular interstitium, decreases testicular proliferative activity, and promotes testicular apoptosis, which may be one of the mechanisms of biliary cirrhosis-related hypogonadism.

Secondary biliary cholestasis promotes testicular macrophage infiltration and autophagy in rats.

PROBLEM: Cholestasis can cause translocation of gut bacteria, and endotoxemia, and systemic inflammation. Now, little is known about the effects of cholestasis on the testicular inflammation and autophagy. METHODS: A rat biliary cholestasis model caused by common bile duct ligation (CBDL), together with biliary decompression (choledochoduodenostomy), was used. RESULTS: The magnitude of MCP-1 expression and CD68(+) macrophage infiltration within testes was progressively up-regulated in rats along with increasing duration of CBDL and was maintained at relatively high level in rats with biliary decompression. The large up-regulation of testicular ATG-12, LC3II, and autophagic vacuoles was found with the extending duration of CBDL and kept at 5 weeks following biliary decompression. The autophagic contents were a large accumulation of mitophagy in testes in rats with CBDL, and cytosol components in rats with biliary decompression. CONCLUSION: Secondary biliary cholestasis can promote inflammatory reaction and the activation of mitophagy and autophagy in testes.

[Effect of spearmint oil on lipopolysaccharide induced emphysema-like changes and expression of matrix metalloproteinase-9].

OBJECTIVE: To investigate the effect of spearmint oil on emphysema-like changes and the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta(IL-1beta), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-9) in lipopolysaccharide (LPS) treated rats. METHOD: Emphysematous changes model was induced by intratracheal instillation of LPS once a week for up to 8 weeks in rats. Rats were divided into control, dexamethasone (0.3 mg x kg(-1)), and spearmint oil (10, 30,100 mg x kg(-1)) groups. Each group was treated with saline, dexamethasone, and spearmint of oil respectively for 4 weeks. Then total and different white blood cell counts in bronchoalveolar lavage fluid(BALF) were carried out. The pathologic changes of lung tissue such as alveolar structure, airway inflammation, and goblet cell metaplasia were observed by HE and AB-PAS staining. Expression of TNF-alpha, IL-1beta, TIMP-1 and MMP-9 were measured. RESULT: Both spearmint and dexamethasone decreased the destruction of pulmonary alveolus. The total and different white blood cell counts in BALF including neutrophile and lymphocyte of spearmint oil 100 mg x kg(-1) and dexamethasone group were significantly reduced, and the goblet cell metaplasia was also inhibited. Dexamethasone had inhibitory effect on the expression of TNF-alpha, IL-1beta, TIMP-1 and MMP-9. Spearmint oil 30, 100 mg x kg(-1) significantly reduced TNF-alpha and IL-1beta respectively. Spearmint oil 10, 30 and 100 mg x kg(-1) had no effect on the expression of TIMP-1, but could decrease the expression of MMP-9 significantly in lung tissues. CONCLUSION: Spearmint oil has protective effect on rats with emphysematous changes, since it improves alveolar destruction, pulmonary inflammation, and goblet cell metaplasia. The mechanism may include reducing TNF-alpha, IL-1beta content and inhibiting overexpression of matrix metalloproteinase-9 in lung tissues.

[Identification of the active material of anti-hepatic fibrosis from Amydae Carapax].

OBJECTIVE: To identify the active material of anti-hepatic fibrosis from Amydae Carapax. METHODS: Membrane separation technology was adopted to screen active fraction in Amydae Carapax, and the active components were isolated from the active fraction using gel chromatography and high performance liquid chromatography. The purified active components in Amydae Carapax were further analyzed using 4700 series time-of-flight mass spectrometer. RESULTS: Proteins and peptides of Amydae Carapax with molecular weight less than 6000 were proved to have biological activity. 8 components (Bj1-Bj8) were isolated from the active fraction. Bj4, Bj6 and Bj7 were screened as active components. Bj7 was further purified, resulting in 7 components (Bj701-Bj707). Bj704 and Bj707 showed significant biological activity. Mass spectrometry showed three molecular ion peaks with highest abundance, i.e. m/e 526, 542 and 572, i.e. m/e 526, 542 and 572, in Bj707 -A The amino acid sequences of above three peptide compounds were NDDY (Asn-Asp-Asp-Tyr), NPNPT (Asn-Pro-Asn-Pro-Thr), and HGRFG (His-Gly-Arg-Phe-Gly), respectively. And M572 was the most abandunt components. CONCLUSION: Three active peptide compounds of anti-hepatic fibrosis of Amydae Carapax were identified.

[Effect of QTY06 on lipopolysaccharide-induced chronic airway inflammation and MUC5ac secretion in rats].

OBJECTIVE: To investigate the effect of synthetic drug QTY06 on chronic airway inflammation and mucoprotein expression induced by intratracheal (i.t) instillation of lipopolysaccharide (LPS). METHODS: Chronic airway inflammation was induced by i.t instillation of LPS in rats. Phospholipids content and the number of leucocytes in bronchoalveolar lavage fluid (BALF), pathological and immunochemical changes were examined 3 weeks after LPS instillation. The effect of QTY06 on chronic airway inflammation was observed. RESULT: After treatment with QTY06, phospholipids in BALF was significantly increased, and the percentages of neutrophils and lymphocytes were decreased as well as the total number of leucocytes. Compared with the model group, pathological examination showed that tracheitis, bronchitis and pulmonary interstitial inflammation in QTY06 groups were significantly attenuated; epithelial damage was alleviated, infiltration of inflammatory cells reduced and the number of goblet cells decreased. QTY06 significantly decreased MUC5ac expression in trachea and bronchiole epithelium, and reduced the optical density and mucins area (%) as detected by image analysis in rats with chronic airway inflammation. CONCLUSION: QTY06 can reduce and inhibit the chronic airway inflammation induced by LPS in rats, and increase the content of phospholipids in pulmonary surfactant and inhibit the hypersecretion of airway mucins.

[Effect of Spearmint oil on inflammation, oxidative alteration and Nrf2 expression in lung tissue of COPD rats].

OBJECTIVE: To investigate the effect of Spearmint oil on inflammation, oxidative alteration and Nrf2 expression in rats with chronic obstructive pulmonary disease(COPD). METHODS: COPD model was induced by intratracheal instillation of Klebsiella pneumonia and lipopolysaccharide (LPS) for 12 weeks in rats, and COPD rats were treated with Spearmint oil for 3 weeks. After COPD was induced, the pathological changes, changes in leucocyte number in blood and bronchoalveolar lavage fluid (BALF), MDA in lung homogenate and Nrf2 expression were observed. The effects of Spearmint oil on these changes were determined. RESULT: Spearmint oil 100 mg*kg(-1)significantly reduced leucocyte numbers in BALF, and attenuated bronchiolitis, pulmonary interstitial inflammation and inflammation cell infiltration. Spearmint oil 30-300 mg*kg(-1)decreased the destruction of pulmonary alveolus and the thickness of bronchioles walls, and inhibited goblet cell proliferation. Spearmint oil significantly reduced MDA in lung homogenate, and decreased the expression of Nrf2 protein in lung tissues. CONCLUSION: Spearmint oil has protective effect on lung injury in COPD rats, since it improves pulmonary inflammation,oxidative alteration, and enhances Nrf2 protein expression.

[Effect of mucosal protective on the quality of gastric ulcer healing].

OBJECTIVE: To explore the mucosal protective effect on the quality of gastric ulcer healing. METHODS: Gastric ulcers were induced in male rats by serosal application of acetic acid. Rats were gavaged for 14 days with saline, omeprazole (OME), teprenone (TEP) and TEP plus OME starting 3 days after ulcer induction. Then the tissues and blood samples were obtained and measured. RESULT: The lower ulcer index (UI) and increased ulcer inhibition rate were observed in OME and OME+TEP groups. In TEP and OME+TEP groups, restored mucosa thickness increased, cystically dilated glands decreased, microvessels in connective tissue increased, the secretion of mucus, hexosamine, PGE(2), bFGF were enhanced, the expression of EGFR was increased. CONCLUSION: TEP can improve the quality of gastric ulcer healing, when combined with OME,the effect is more marked.

[The expression of TLR4 in rat acute lung injury induced by lipopolysaccharide and the influence of Eucalyptus globulus oil].

OBJECTIVE: To observe the distribution of toll-like receptor 4 (TLR4) in rats' respiratory tract. To study the influence of LPS and Eucalyptus globulus oil on the distribution of TMR4. METHOD: The Sprague-Dawley rats were intratracheally instilled with lipopolysaccharide (LPS,2 mg x kg(-1) per day) for two days to induce acute lung injury. The rats were sacrificed at 72 hours after LPS instillation. Lung morphology was studied. Leukocytes in Bronchoalveolar lavage fluid (BALF) were measured and TLR4 were detected by immunohistochemistry. RESULT: The result of immunohistochemistry showed that TLR4 distributed widely in common rats' respiratory tract. In the group of acute lung injury, the number of leucocyte in BALF was increased apparently, the inflammation in bronchus and bronchioles was more apparently than that of the control group in morphology. And the expression of TLR4 was reinforced in main bronchus and bronchioles. In the group of E. globules oil (300 mg x kg(-1)), the leucocyte number was decreased apparently in BALF, the inflammation was lightened and the expression of TLR4 decreased as compared with the group of models. CONCLUSION: The expression of TLR4 distributes widely in rats' respiratory tract. The stimulation of LPS can reinforce the expression of TLR4. The E. globules oil can reduce the increase of TLR4 induced by LPS in bronchioles.

Effects of combination of irbesartan and perindopril on calcineurin expression and sarcoplasmic reticulum Ca2+-ATPase activity in rat cardiac pressure-overload hypertrophy.

AIM: To observe effects of angiotensin (Ang) II receptor antagonist (AT1) irbesartan and angiotensin-converting enzyme (ACE) inhibitor perindopril on rat myocardium calcineurin expression and sarcoplasmic reticulum Ca(2+)-ATPase activity in the model of pressure-overload cardiac hypertrophy. METHODS: Forty male adult Sprague Dawley rats were divided into 5 groups. One group was treated by sham operation; four groups were myocardium hypertrophy cases caused by banding aortic above renal artery. Drugs were given one week after operation. Group 1: sham group, rats (n=8) were gavaged with normal saline 2 ml/(kg.d) (ig); Group 2: control group, rats (n=8) were treated with normal saline 2 ml/(kg.d) (ig); Group 3: rats (n=8) were given perindopril 2 mg/(kg.d) (ig); Group 4: rats (n=8) were treated with irbesartan 20 mg/(kg.d) (ig); Group 5: rats (n=8) were given irbesartan 20 mg/(kg.d) plus perindopril 2 mg/(kg.d) (ig). Morphometric determination, calcineurin expression and sarcoplasmic reticulum Ca(2+)-ATPase activity were done at the end of 6 week of drug intervention. Expression of calcineurin in myocardium was detected by immunohistochemistry. RESULTS: Left ventricular mass index (LVMI), transverse diameter of myocardial cell (TDM), calcineurin activity were remarkably decreased after drug intervention and this decrease was most remarkable in the combination drug therapy group. Sarcoplasmic reticulum Ca(2+)-ATPase activity was increased after drug intervention, especially in the combined drug therapy group. Calcineurin expression in myocardium was remarkably decreased after drug intervention. LVMI was positively correlated with TDM and calcineurin, negatively correlated with sarcoplasmic reticulum Ca(2+)-ATPase. CONCLUSION: These data suggest that irbesartan and perindopril inhibit cardiac hypertrophy through the increased activity of sarcoplasmic reticulum Ca(2+)-ATPase and decreased expression of calcineurin. Their combination had better effects on regressing of ventricular hypertrophy.

[Forensic pathological significance of immunohistochemical study with CX43 in rats on early myocardial ischaemia].

OBJECTIVE: To explore one of evidence for pathologic diagnosis of early myocardial ischaemia. METHODS: Rats were ligated of the left coronary artery according to a previously documented technique, and heart tissue was sampled at different ischaemia time. The expression of CX43 in myocardial cell was detected by Immunohistochemistry. RESULTS: It is showed that the distribution and amount of CX43 positive staining in each group of the myocardial ischaemia was different from that of the control group. CONCLUSION: The changes of CX43 detected by Immunohistochemical methods may be helpful for the diagnosis of early myocardial ischaemia, but further pathologic investigation and research is necessary.

[Mechanisms of muscovite on gastric mucosal protective effect].

OBJECTIVE: To explore the mechanisms of muscovite gastric mucosal protective effect. METHOD: Rat model of chronic gastritis were used. After gastric mucosal injury was induced, the rats were divided into 6 groups and were treated with different drugs. 2 weeks later, the tissue and blood samples were obtained and measured. RESULT: The general conditions, the observations under macroscopy, microscope and electron microscope of the middle and high dose of muscovite groups resembled those of the normal group. Their PH levels were higher than those of the model group, and the rates of intestinal metaplasia were lower, but the PGE2 level of the middle dose of muscovite group was the highest. CONCLUSION: Muscovite can be adsorbed on the surface of the gastric mucosa. It has gastric mucosal protective effect by improving excretion of mucus and synthesis of PGE2 in gastric mucosa, restraining gastric acid, reversing of intestinal metaplasia and decreasing inflammation cells.

Intrathymic inoculation of liver specific antigen alleviates liver transplant rejection.

OBJECTIVE: To study the effects of liver specific antigen (LSA) on liver allotransplantation rejection. METHODS: Orthotopic liver transplantation was performed in this study. Group I: syngeneic control (Wistar-to-Wistar); Group II: acute rejection (SD-to-Wistar). Group III: thymic inoculation of SD rat LSA day 7 before transplantation. The observation of general condition and survival time, rejection grades and the NF-kappaB activity of splenocytes were used to analyze severity of acute rejection and immune state of animals in different groups. RESULTS: The general condition of group I was fair post transplantation with no sign of rejection. All recipients of group II died within days 9 to 13 post transplantation with median survival time of 10.7 +/- 1.37 days. As for group III, 5 out of 6 recipients survived for a long period with remarkably better general condition than that of group II. Its rejection grades were significantly lower than group II (P<0.05). NF-kappaB activity was only detected in group I between days 5 and 7 after transplantation, whereas high activity of NF-kappaB was detected at all points in group II and low NF-kappaB activity was detected in group III which was significantly lower than that of group II (P<0.05). CONCLUSIONS: LSA is an important transplantation antigen directly involved in the immunorejection of liver transplantation. Intrathymic inoculation of LSA can alleviate the rejection of liver allotransplantation, grafts survive for a period of time thereby, allowing a novel way to liver transplantation immunotolerance.

Inhibitory effect of tea polyphenols on transforming growth factor-beta1 expression in rat with cyclosporine A-induced chronic nephrotoxicity.

AIM: To investigate the inhibitory effect of tea polyphenols (TP) on the transforming growth factor-beta1 (TGF-beta1) expression in rat model of cyclosporine A (CsA)-induced chronic nephrotoxicity. METHODS: The rat model of CsA-induced chronic nephrotoxicity was used, 4 groups of rats were respectively treated with vehicle (0.1 mL/kg/d sc), TP (80 mg/kg/d ig), CsA (15 mg/kg/d sc) and TP plus CsA (CsA 15 mg/kg/d sc+TP 80 mg/kg/d, ig). At the end of day 28 of treatment, serum and urine are analyzed for creatinine clearance, kidney tissue for pathologic analysis. The TGF-beta1 mRNA and its protein expression were detected by RT-PCR, immunohistochemistry, and Western blot. RESULTS: CsA-treated rats had increased renal expression of TGF-beta1 mRNA and its protein, compared with the vehicle- or TP-treated controls. The renal function and interstitial fibrosis were ameliorated and renal expression of TGF-beta1 mRNA and its protein was decreased in animals treated with CsA plus TP, compared with animals treated with CsA alone (P<0.05). CONCLUSION: TP significantly inhibits renal expression of TGF-beta1 in rat model of cyclosporine-induced chronic nephrotoxicity, suggesting that the decreased renal expression of TGF-beta1 exerted by TP is one of mechanisms to protect renal function and tissue structure.

Inhibitory effect of tea polyphenols on renal cell apoptosis in rat test subjects suffering from cyclosporine-induced chronic nephrotoxicity.

OBJECTIVE: To investigate the inhibitory effect of tea polyphenols on renal cell apoptosis in rat test subjects suffering from cyclosporine A (CsA)-induced chronic nephrotoxicity. METHODS: Four groups of rats with CsA-induced chronic nephrotoxicity were respectively treated with vehicle olive oil, tea polyphenols, CsA and tea polyphenols plus CsA. At the end of the 28th day of treatment, 24 hours urine and blood samples were obtained, and the animals were then sacrificed. The serum and urine samples were analysed for creatinine clearance, and kidney tissue was used for pathologic analysis of renal tubular injury and interstitial fibrosis. The TUNEL assay, apoptosis-related enzyme caspase-3 mRNA detected by RT-PCR, and its enzymatic activity were analysed for the possible detections of cell apoptosis. RESULTS: CsA-treated rats displayed increased apoptosis of the tubular and interstitial cells, in comparison with vehicle-treated controls (18.3 +/- 4.6 vs 4.8 +/- 1.3 cells/mm(2), P < 0.05). In comparison with animals treated by CsA, animals treated with CsA plus tea polyphenols demonstrated significantly improved levels of creatinine clearance (0.12 +/- 0.03 vs 0.22 +/- 0.02 ml.min(-1).100 g(-1) body weight, P < 0.05), tubular injury (2.29 +/- 0.43 vs 1.42 +/- 0.26, P < 0.05), and interstitial fibrosis (2.83 +/- 0.20 vs 1.46 +/- 0.19, P < 0.05), and showed a statistically significant decrease in tubular and interstitial cell apoptosis (18.3 +/- 4.6 vs 7.7 +/- 2.1 cells/mm(2), P < 0.05). The expression of caspase-3 mRNA and caspase-3 activity was significantly higher in the CsA-treated group than that of the CsA plus tea polyphenols (TP)-treated group (P < 0.05). CONCLUSION: These results suggested that tea polyphenols significantly inhibits apoptosis of the tubular and interstitial cells in rats with cyclosporine-induced chronic nephrotoxicity, and that tea polyphenols may be useful to prevent CsA-associated kidney toxicity.

Correlated expression of inducible nitric oxide synthase and P53, Bax in benign and malignant diseased gallbladder.

Our goal has been to investigate the expression and correlated significance of inducible nitric oxide synthase (iNOS) and P53, Bax in benign and malignant gallbladder diseases. We detected the expression of iNOS, P53 and Bax in the gallbladder wall by SP immunohistochemistry in 16 cases of chronic cholecystitis, 11 cases of chronic cholecystitis with adenomyoma and 24 cases of gallbladder adenocarcinoma. The percentage of positively marked tumor cells was counted under microscope and the intensity of immunoreactivity was graded. SPSS10.0 statistical software was applied for statistical analysis. In this study, we found that: (1) Both benign and malignant diseased gallbladder wall expressed iNOS and Bax. Compared to benign diseased gallbladders, their expression in adenocarcinoma was decreased (p < 0.05), P53 was expressed strongly only in nuclei of adenocarcinoma cells of some cases. (2) In benign and malignant diseased gallbladders, iNOS expression was related positively to Bax (p < 0.01), the expression of P53 and Bax had a negative relationship (p < 0.01). The results suggested that both chronic cholecystitis and chronic cholecystitis with adenomyoma carry the risk of becoming malignant, especially the latter. NO is an important mediated molecule in cancer, there are intimate relationships between gallbladder cancer and apoptosis.

[The effect of an antioxidant tea polyphenols on cell apoptosis in rat model of cyclosporine-induced chronic nephrotoxicity].

OBJECTIVE: To investigate the effect of tea polyphenols on cell apoptosis in rat model of cyclosporine-induced chronic nephrotoxicity. METHODS: Four groups of animals in rat model of cyclosporine-induced chronic nephrotoxicity were respectively treated by olive oil (n = 6), tea polyphenols (TP, n = 6), cyclosporine A (CsA, n = 8) and TP plus CsA (n = 8). At the end of 28th day of treatment, all animals were sacrificed and blood was analyzed for blood serum creatinine and creatinine clearance, kidney tissue for pathologic analysis. The TUNEL assay, caspase-3 mRNA expression detected by reverse transcription-polymerase chain reaction (RT-PCR) and caspase-3 activity were used for the analysis of cell apoptosis. RESULTS: CsA plus TP ameliorated the CsA-induced decrease of renal function and interstitial fibrosis. There was a significant increase in the number of apoptosis-positive cells in the CsA-vs-CsA plus TP-treated group at four weeks (18.9 +/- 3.3 vs. 7.7 +/- 1.4, P < 0.05). The expression of caspase-3 mRNA and caspase-3 activity of CsA-treated group was significantly higher than that of CsA plus TP-treated group (P < 0.05). CONCLUSION: These results indicate that antioxidant tea polyphenols significantly inhibit apoptosis of tubular and interstitial cells in rat model of chronic cyclosporine-induced nephrotoxicity, and suggest that the decrease of cell apoptosis exerted by tea polyphenols may be one of mechanisms to protect renal function and tissue structure.