[Effect of inhibiting the activity of double-stranded RNA-dependent protein kinase in sepsis mice].

Objective: To observe the effects of 2-aminopurine (2-AP), a double-stranded RNA-dependent protein kinase (PKR) inhibitor, on organ function, plasma inflammatory factor expression and 7 days mortality in sepsis mice induced by cecal ligation puncture (CLP). Methods: Forty specific specific pathogen free C57BL/6 mice were randomly divided into sham group (n=10), CLP group (n=10), CLP+2-AP group (n=10) and 2-AP group (n=10). CLP was used to establish sepsis mice models.Peripheral blood serum was collected 24 hours after operation, alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (Cr), blood urea nitrogen (BUN) and inflammatory factors (IL-1ß, IL-10 and TNF-α) were detected; peripheral blood and peritoneal lavage fluid were taken for bacterial clearance detection. Another 60 C57BL/6 mice were selected to observe the 7-day survival rate according to the above groups (n=15). Independent sample t test was used to compare the measurement data between groups. Results: The levels of ALT, AST, Cr and BUN in CLP Group and CLP+2-AP group were significantly higher than those in sham group (all P<0.001). The levels of ALT and AST in CLP+2-AP group were significantly lower than those in CLP Group (t=27.88, 11.33, both P<0.001); the levels of Cr and BUN in CLP+2-AP group were significantly lower than those in CLP Group (t=11.02, 7.15, bothP<0.001). Compared with sham group, the levels of pro-inflammatory (IL-1ß and TNF-α) and anti-inflammatory (IL-10) cytokines in CLP group were significantly higher (all P<0.001); the levels of IL-1ß and IL-10 in CLP+2-AP group were significantly lower (all P<0.001), but the levels of TNF-α in CLP+2-AP group were not significantly lower (P=0.33). The 7-day survival rate was 100% in sham group, 13.3% in CLP+2-AP group, 86.7% in 2-AP group and 20.0% in CLP+2-AP group. Inhibition of PKR activation slightly improved the trend of 7-days survival rate of CLP model mice (analysis by mantel Cox test, χ(2)=0.0012, P=0.97). Conclusion: In sepsis mice model, inhibition of PKR activity can reduce the expression of inflammatory factors in plasma, decrease bacterial load in blood and abdominal cavity, and protect organ function, which could suggest that inhibition of PKR activity has potential application in sepsis treatment.

[Serum procalcitonin in patients with pulmonary infection and central nervous system injury].

Objective: To evaluate the influence of serum procalcitonin in the diagnosis and treatment of pulmonary infection in patients with central nervous system injury. Methods: From October 2014 to February 2017, a retrospective study was performed. A total of 1 852 patients were screened in Department of Intensive Care Unite, First Affiliated Hospital of Sun Yat-sen University.Among them, 173 patients were identified with different kinds of infection. Finally, a total of 42 patients with pulmonary infection were enrolled. The clinical data of patients with pulmonary infection and central nervous system (CNS) injury was collected. A univariate and multivariate regression analysis was performed to study the correlation of serum procalcitonin (PCT) with clinical symptoms and signs of the pulmonary infection, body temperature(T), white blood cell count (WBC), percentage of neutrophils (NEU) and the severity of the pulmonary infection (CPIS). The relationship of serum PCT with type of CNS injury, GCS, and exogenous glucocorticoid was further studied. Results: During the period of pulmonary infection, the peak PCT was 0.83 (0.29, 2.79) µg/L and the CPIS was 5.50 (5.00, 7.00). In 9 of 42 patients, the peak PCT was less than 0.25 µg/L. In 7 of 42 patients, the peak PCT was ranged from 0.25 to 0.5 µg/L. In 12 of 42 patients, PCT was ranged from 0.5 to 2 µg/L. Only 10 patients had a PCT 2-10 µg/L and 4 patients had a PCT more than 10 µg/L. There is no correlation between serum PCT and body temperature, white blood cell, percentage of neutrophils and CPIS. There was no significant differences in patients with PCT<0.5 or ≥0.5 µg/L regarding the body temperature, white blood cell, percentage of neutrophils and CPIS. However, serum PCT in patients with pulmonary infection had independent correlation with the post CNS injury day (ß=0.17, 95% CI (0.02, 0.32), P<0.05). The serum PCT was 1.26 (0.47, 2.7) µg/L and 29.41% patients with a PCT less than 0.5 µg/L within 3 days post CNS injury. Serum PCT level was 0.23 (0.16, 0.39) µg/L, and 77.78% patients with a PCT less than 0.5 µg/L at day 4 to day 7 post-injury. The PCT level was 0.52 (0.33, 1.12) µg/L, and 44.44% patients with a PCT less than 0.5 µg/L at day 8 to day 14. The PCT was 3.26 (2.07, 12.40) µg/L, and no patient with a PCT less than 0.5 µg/L after day 15 post-injury. There were no significant relationship found between serum PCT level and type of the disease and surgery, GCS, and use of exogenous glucocorticoid. Conclusions: Serum PCT had no significant increase and was not able to be used in guiding the antibiotics use in patients with CNS injury and pulmonary infection.

Regulatory T cells in chronic hepatitis B patients affect the immunopathogenesis of hepatocellular carcinoma by suppressing the anti-tumour immune responses.

Chronic hepatitis B (CHB) virus hepatitis B virus (HBV) infection is the key cause of hepatocellular carcinoma (HCC) in Asians. Recent studies have shown that levels of CD4(+)CD25(+) regulatory T cells (Tregs) were increased and were linked to an impaired immune response in patients with CHB. Evaluating whether Tregs are involved in the progression of CHB to HCC will provide insight into the immunopathogenesis of HCC. In the present study, we showed that circulating and liver-residing Tregs increased in CHB (n = 15) and HCC (n = 49) patients, particularly in the peripheral blood of HCC patients with HBV infection (n = 29). The increased Tregs in CHB patients suppressed the specific immune response induced by not only HBV antigen, but also by HCC tumour antigen. When peripheral blood mononuclear cells (PBMC) were co-cultured with human hepatoma cell lines that are stably transfected with HBV (HepG2.2.15), CD4(+)CD25(+) Treg populations increased and upregulated the expression of forkhead box P3 transcriptional regulator (FoxP3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and glucocorticoid-induced tumour necrosis factor (TNF) receptor family gene (GITR). In contrast, PBMCs co-cultured with HepG2 cells (the parental cell line of HepG2.2.15) did not. CD4(+)CD25(+) Tregs isolated from PBMCs that were co-cultured with HepG2.2.15 cells also had a greater suppressive ability with respect to the tumour antigen-specific immune response induced by NY-ESO-1 or MAGE-A3 compared with CD4(+)CD25(+) Tregs isolated from PBMCs co-cultured with HepG2 cells. The results offer evidence that the expansion of CD4(+)CD25(+) Tregs and the enhancement of the suppressor function of CD4(+)CD25(+) Tregs induced by HBV infection-related factors could suppress the anti-tumour immune response to HCC tumour antigen and inhibit tumour immuno-surveillance against HCC, which may be involved in the immunopathogenesis from CHB to HCC.

[A study of the protective mechanism of hepatic stimulator substance against experimental acute liver failure in mice].

In our previous work we have demonstrated a protective effect of hepatic stimulator substance (HSS) on experimental hepatic injury in mice. The underlying mechanism of this effect was further investigated with the following results: (1) HSS could significantly increase the CCl4-induced decrease of membrane fluidity of hepatic plasmalemma, mitochondria and microsomes to the control level. (2) HSS could decrease the liver melondialdehyde contents of CCl4-intoxicated mice. (3) HSS could restore the liver glutathione contents lowered by CCl4-intoxication. (4) HSS stimulated regeneration of liver, enhanced DNA synthesis of hepatocytes and increased 3H-TdR incorporation into DNA. According to the results mentioned above, we proposed that HSS should have an antioxidative effect on the hepatic membrane lipid peroxidation which was increased by free radicals produced by CCl4. In addition, HSS might increase antioxidative ability of hepatocyte and stimulate the proliferation of hepatocytes. These protective mechanisms might act in a concordant manner.

[Effect of carbon tetrachloride intoxication on leakage of intracellular potassium in regenerating rat hepatocytes in vitro].

Leakage of intracellular potassium and GPT in normal rat hepatocytes exposed to 10, 15 and 20 mmol/L of carbon tetrachloride (CCl4) in vitro was determined at 5, 10, 15 and 20 min after cell intoxication. Both of the intracellular potassium and GPT were clearly dose- and time-dependent with CCl4, but the leakage of intracellular potassium is a more sensitive index to indicate cell injury than the later. The percent change of intracellular potassium in regenerating rat hepatocytes 20 min after CCl4 (15 mmol/L) intoxication was markedly reduced than that in normal rat. This finding indicates that regenerating hepatocytes are resistant to CCl4 hepatotoxicity in vitro, and its mechanism presumably is due to a higher stability of regenerating liver cell membrane.

[Protective effect of hepatic stimulator substance against experimental acute liver failure in mice].

A hepatic stimulator substance (HSS) was extracted from the liver of male weanling SD rats according to the method of LaBrecque. The mice were injected with carbon tetrachloride or D-galactosamine to induce hepatic injuries and the protective effect of HSS on thus induced hepatic damage was investigated. The results were as follows: (1) HSS could suppresses the elevation of sGPT and sGOT induced by carbon tetrachloride intoxication in a dose-dependent manner. (2) Hepatic histological findings indicated that the degree of CCl4 or D-galactosamine-induced hepatic lesions could be lessened by HSS. (3) CCl4-induced reduction of hepatic mitochondrial succinic dehydrogenase activity could be restored by HSS. (4) Insulin-glucagon enhanced the survival of D-galactosamine intoxicated mice and stimulated hepatocyte proliferation, thus showing less pronounced hepatic damage.

[Effect of human hepatic stimulator substance on intracellular calcium and potassium homeostasis in CCl4-intoxication dissociated hepatocytes].

The human hepatic stimulator substance (hHSS) was extracted from abortive fetal liver according to the method of LaBrecque. The intracellular free calciumion was measured by fluorescent probes Fura-2/AM. Rat hepatocytes were isolated from liver by the method of Seglen and intoxicated by CCl4 vapour, then the changes of [Ca2+]i, the leakage of intracellular K+, ALT and viability of hepatocytes were observed. The results were as follows: The hHSS does exist in the human fetal liver, hHSS could increase the viability of intoxicated hepatocytes, maintain the intracellular calcium homeostasis, and decrease the leakage of intracellular potassium and ALT into the culture medium. These results indicate that hHSS could protect hepatocytes against CCl4 through maintaining calcium homeostasis, preventing potassium leakage and sustaining stability of hepatocyte polasmalemma.

[Effects of calcium ion on contractile response of guinea-pig gallbladder muscle strip to agonists].

The effects of calcium ion on the contractile response of gallbladder muscle strip (GBMS) in guinea pigs to acetylcholine, carbachol and caerulein were investigated by method of isotonic contraction and by RIA of cGMP and cAMP in the tissue. It was shown that the contractile response of GBMS to these agonists was stronger in Krebs solution than that in the calcium-free solution. In calcium-free solution, EDTA-zero calcium solution or verapamil-Krebs solution, the contractile response of GBMD was decreased to about 30% of the control (P less than 0.01). Verapamil significantly decreased carbachol induced enhancement of cGMP and cAMP of gallbladder tissue (P less than 0.01 or 0.05). The results suggest that both extra-and intra- cellular calcium take part in the contractile response of GBMS to agonists.

[Participation of dopamine on the muscarinergic inhibitory effect of substance P on gastric myoelectric activity and motility].

Our previous study showed that microinjection of substance P (SP) into caudate nucleus inhibits gastric myoelectric fast wave and gastric motility, an effect mediated by muscarinic receptor. The present investigation showed that this effects of SP could be blocked by coinjected SP antiserum or SP antagonist [Arg6, D-Trip7,9, MePhe8]-SP6-11 or D2 dopamine antagonist haloperidol. In addition, microinjection of dopamine (DA) into caudate nucleus could also inhibit gastric fast wave and motility, an effect again being blockable by coinjected DA antagonist haloperidol or atropine. Thus, it appears that the muscarinergic inhibitory effect of SP on gastric fast wave and motility is mediated by D2 dopamine receptor.

[A study of regenerating liver protects against carbon tetrachloride injury in rat].

In our previous study we had demonstrated that regenerating liver of rats had an ability to resist CCl4 injury. In this paper, the underlying mechanism was further investigated. Hepatic stimulator substance from regenerating liver (rHSS) at different time after partial (68%) hepatectomy was extracted and assayed for its biological activity by using 3H-thymidine. The activity is approximately seven-fold as compared with the control. Then, rHSS was given to rats to observe its effect against CCl4 injury both in vivo and in vitro. The results were as follows: rHSS decreases the mortality of CCl4 intoxicated rats and suppresses the elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) induced by CCl4 in vivo. Also rHSS increases the cell viability and decreases the leakage of intracellular ALT of hepatocytes poisoning by CCl4. The above mentioned results suggest rHSS is an important mechanism for regenerating liver to protect against CCl4 poisoning.

[Protective effect of hepatocyte growth factor on hepatocyte poisoning by carbon tetrachloride and related gene expression in rats].

Hepatocyte growth factor (HGF), as a pleiotropic factor, plays important roles in organ regeneration, organogenesis and morphogenesis, but few reports on protective effect of HGF on injured liver are available. The aim of this work was to investigate protective effects of recombinant human HGF (rhHGF) on intoxicated hepatocytes induced by carbon tetrachloride (CCl4). The results are as follows: (1) rhHGF (5 ng/ml) could significantly increase the viability of hepatocytes intoxicated by CCl4 (15 mmol/L), decrease the leakage of intracellular alanine transaminase (ALT) and potassium ions into the culture medium; (2) the combination of rhHGF (5 ng/ml) and epidermal growth factor (EGF) (50 ng/ml) could increase the viability further, decrease the leakage of intracellular ALT and potassium ions of hepatocytes; (3) high expression of HGF and its receptor/c-met gene in regenerating liver after partial hepatectomy and CCl4 (50%, 2.5 ml/kg bw) poisoning were examined respectively by reverse transcription-polymerase chain reaction in rats. The results showed that rhHGF could protect hepatocytes against CCl4 through preventing intracellular ALT and potassium ion leakage, thus suggesting a synergistic effect of rhHGF and EGF on cytoprotection, and high expression of HGF and its receptor/c-met mRNA in the liver treated with PH and CCl4 poisoning. The present study suggests that the expression of HGF and its receptor gene plays an important role in liver regeneration and repair.

Potentiated effect of vagal stimulation and hormonal agents on pancreatic exocrine secretion in dogs.

Chronic experiments were made on eighteen dogs with Thomas pancreatic fistula and gastric fistula. Both in chronic and acute experiments the degenerated right cervical vagus nerve was stimulated by electrical shock and the endogenous secretin and CCK was released by means of duodenal acidification (D. A.). The exogenous secretin, atropine and lidocaine were infused to analyse the interrelationship of neurohormones in the pancreatic exocrine secretion. The results were as follows. In chronic experiments the pancreatic secretory latency was shorter and volume larger than that in acute experiments induced by D. A. The difference is very significant (P less than 0.001). Both vagotomy and atropine significantly inhibited pancreatic secretion induced by D. A. (P less than 0.01). Lidocaine infused into duodenum inhibited pancreatic secretion induced by D. A. as well. When vagal stimulation was combined with D. A., either simultaneously or successively, the pancreatic secretion was increased more than the additive sum obtained by separate action of vagal stimulation and D. A. or secretin. In view of the above-mentioned facts, the author suggests that vagal impulses combined with D. A. or secretin act on the pancreatic exocrine secretion, and that the interaction of nerve and hormones appear to be mutually potentiated.

[Control of growth and expression of protooncogenes in regenerating liver].

There are many humoral factors involved in the control of growth in regenerating liver. The complete hepatocyte mitogens such as hepatocyte growth factor (HGF), hepatic stimulator substance (HSS) can strongly stimulate hepatocyte DNA synthesis and mitosis. The hepatocyte growth inhibitors such as transforming growth factor beta 1 (TGF beta 1), however, do not stimulate DNA synthesis, but inhibit EGF mitogenesis. In addition, the comitogens such as norepinephrine and insulin are necessary to regulate the growth of regenerating liver. It has become clear that the hepatocyte proliferation and protooncogenes are linked closely. Some protooncogenes can express specifically as markers in the different phases of the cell cycle and in hepatocytes that enter the cell cycle (G0 to G1 transit) and continue to progress.