Effect And Possible Mechanism Of Costimulatory Molecule B7-H3 In Acute Pancreatitis

Section 1: Effect and possible mechanism of anti-B7-H3 monoclonal antibody in acute pancreatitisObjective: To explore the effect and possible mechanism of anti-B7-H3 monoclonal antibody(m Ab) in acute pancreatitis.Methods: Mice were randomly divided into four groups: control group, AP group, Ig G treatment group and anti-B7-H3 m Ab treatment group. AP was induced in mice by administration of intraperitoneal injections of L-arginine. B7-H3 m Ab was administered to the mice before injections of L-arginine. The first L-arginine injection is defined as 0 hour. The blood, pancreas and lung tissues of mice were collected at 2d, 3d, 4d. Expressions of B7-H3 protein were detected in the pancreas tissues of the control and AP group by Western blot. Serum activities of amylase and lipase were tested. Pathological changes of pancreas and lung tissues were valued by HE staining. Serum levels of tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and IL-1β were detected by ELISA. Expressions of phospho NF-κB protein were detected in the pancreas tissues by Western blot.Results: B7-H3 was undetectable in normal murine pancreas, while obviously increased with times in AP group. Markedly decreased amylase and lipase activities were observed in anti-B7-H3 m Ab treatment group. Anti-B7-H3 m Ab treatment ameliorated the L-arginine-induced damage of pancreas and lung tissues. The levels of serum TNF-α, IL-6, IL-1β and the expression of phospho NF-κB were significantly increased in L-arginine-induced AP, and decreased in anti-B7-H3 m Ab treatment group.Conclusions: B7-H3 participates in the development of acute pancreatitis, and anti-B7-H3 m Ab ameliorates acute pancreatitis via attenuation of inflammatory response.Section 2: Effect and possible mechanism of anti-B7-H3 monoclonal antibody in coculture model of acinar cells and macrophagesObjective: To explore the effect and possible mechanism of anti-B7-H3 monoclonal antibody(m Ab) in coculture model of acinar cells and peritoneal macrophages.Methods: The isolated pancreatic acinar cells were divided into four groups: control group, L-arginine group, Ig G treatment group, and anti-B7-H3 m Ab treatment group. 5mg/ml L-arginine was administered in L-arginine group. Anti-B7-H3 m Ab or Ig G was administered in anti-B7-H3 m Ab treatment group or Ig G treatment group before administrated L-arginine. Supernatants were collected and detected in levels of TNF-α, IL-6, and IL-1β by ELISA. The isolated peritoneal macrophages were divided into four groups: control group, LPS group, L-arginine group and supernatant group. 1μg/ml LPS, 5mg/ml L-arginine, or supernatant of acinar cells cultured with L-arginine for 24 h was administered. Supernatants were collected and detected in levels of TNF-α, IL-6, and IL-1β by ELISA. The coculture model of acinar cells and macrophages were divided into four groups: control group, AP group, Ig G treatment group, and anti-B7-H3 m Ab treatment group. Levels of TNF-α, IL-6, and IL-1β were detected by ELISA. Expressions of phospho NF-κB in macrophages were detected. CCK-8 was to test pancreas acinar cells viability.Results: In the culture of acinar cells, low levels of proinflammatory cytokines were detected in cell-free supernatant of acinar cells cultured with L-arginine, and these levels were not affected by the anti-B7-H3 m Ab. In the culture of peritoneal macrophages, levels of TNF-α, IL-6, and IL-1β in supernatants of L-arginine group showed no significant difference with control group, while the supernatant group showed significant high levels. Pancreatic acinar cells and peritoneal macrophages were cocultured successfully. The levels of supernatant TNF-α, IL-6, IL-1β and the expression of phospho NF-κB in macrophages were significantly increased in AP group, and decreased in anti-B7-H3 m Ab treatment group. And acinar cells viability was increased in anti-B7-H3 m Ab group.Conclusions: Anti-B7-H3 m Ab ameliorates inflammatory response in macrophages by NF-κB signal pathway.Section 3: The levels and clinical significance of soluble costimulatory B7-H1, B7-H2 and B7-H3 in the peripheral blood of acute pancreatitisObjective: To explore the value of soluble costimulatory B7-H1, B7-H2 and B7-H3 in the development and severity of acute pancreatitis.Methods: Setting 20 healthy persons as control group, the blood samples were collected from 66 AP(24 MAP, 24 MSAP, 18 SAP) patients at 1d, 3d and 7d. The levels of s B7-H1、s B7-H2 and s B7-H3 were measured by the ELISA kits.Results: The levels of s B7-H1 in AP group were higher than control group. s B7-H1 was increased straightly in SAP group and significant difference was detected between 3d and 7d. s B7-H1 of SAP in 7d was significantly higher than MSAP. The levels of s B7-H2 in AP group were lower than control group and decreased with the development of AP. MAP group in 3d had significantly decreased compared with 1d. MAP group had significantly higher than MSAP group in 7d. The levels of s B7-H3 in AP group were increased compared with control group. In SAP group, B7-H3 in 7d was significantly increased. The levels of B7-H3 were increased with the severity of AP, though there was no significant difference compared with MAP and MSAP in 1d. The accurate rate of s B7-H3 in diagnosis of SAP was increased with the disease progression.Conclusions: Dynamic analysis of soluble costimulatory B7-H1, B7-H3 and B7-H2 in the peripheral blood of AP has the value to assess the development and the severity of the disease.