Th17/Treg Dysregulation In The Early Stage Of Severe Hepatitis B And Rebalance After Glucocorticoid Treatment

Part one: The construction and functional identification of the early stage of severe hepatitis animal modelObjective: To construct the animal models with early stage of severe hepatitis. Methods: Different doses of carbon tetrachloride through intraperitoneal injection to build mice models with early stage of severe hepatitis. Different doses of carbon tetrachloride through intraperitoneal injection to build rat models with early stage of severe hepatitis. Different doses of D-galactosamine(D-Gal N) and 1ipopolysaccharide(LPS) through intraperitoneal injection to build rat models with early stage of severe hepatitis. Respectively to observe the liver changes, pathology changes and serum biochemical changes of animal models. Results: 9 mice of 21 died in 36 hours with different doses of carbon tetrachloride. Liver cells swelled below 25 mg/kg dose group, but more than 50 mg/kg dose, there were chunk or large necrotic liver histology. 2 SD rats of 10 died in 240 hours with different doses of carbon tetrachloride. There were hepatocyte swelling and mild necrosis below 50 mg/kg dose, but more than 500 mg/kg dose, there were large necrotic liver histology. 4 SD rats of 12 died in 240 hours with different doses of D-Gal N combined LPS. There were hepatocyte swelling and mild necrosis below 700 mg/kg D-Gal N combined 35 ug/kg LPS dose, but more than 3500 mg/kg D-Gal N combined 175 ug/kg LPS dose, there were large necrotic liver histology. Conclusions: Firstly, the early stage of the severe hepatitis mice models are established with carbon tetrachloride, the carbon tetrachloride dose of 25 mg/kg is appropriate. No deaths occurred in 36 hours, liver histology and serum biochemistry changes also conforms to the early stage of the severe hepatitis. Secondly, the early stage of the severe hepatitis Rat models are established with carbon tetrachloride, the carbon tetrachloride dose of 50 mg/kg is appropriate. No deaths occurred In 240 hours, liver histology and serum biochemistry changes also conforms to the early stage of the severe hepatitis. Thirdly, the early stage of the severe hepatitis Rat models are established with D-Gal N combined LPS, the 350mg/kg D-Gal N combined 17.5ug/kg LPS dose is appropriate. No deaths occurred In 240 hours, liver histology and serum biochemistry changes also conforms to the early stage of the severe hepatitis.Part two: Th17/Treg dysregulation in animal models with early stage of severe hepatitis and the alterations of the Th17/Treg dysregulation after glucocorticoid and thymosin interventionObjective: To study the changes of Th17, Treg and Th17/Treg in animal models with early stage of severe hepatitis and the alterations of these cells after glucocorticoid and thymosin intervention. Methods: The mice blood after removing eye, rats caudal vein blood, liver and spleen tissue homogenate(diluted to 106-109 mononuclear cells/m L) 100 ul was put into test tubes with sodium heparin for the detection of specific immune cells by intracellular cytokine identification using in vitro stimulation and fixation/permeabilization followed by flow cytometry. Results: The early stage of the severe hepatitis animal models had higher Th17, lower Treg and Th17/Treg dysregulation. All early stage of the severe hepatitis rat models had decreases in Th17 levels, increases in Treg, and rebalanced Th17/Treg ratios after glucocorticoid treatment. Animal models occured increase Th17, slightly low Treg after thymosin intervention. Conclusions: All the animal models with early stage of severe hepatitis had Th17/Treg imbalances than normal control. The Th17/Treg imbalances improved after dexamethasone intervention, animal histology improved than no using dexamethasone intervention. Animal models occured increase Th17, slightly low Treg after thymosin intervention, therefore, dexamethasone can improve early severe hepatitis, but thymosin is likely to lead to disease progression.Part three: Th17/Treg dysregulation in the early stage of severe hepatitis B and rebalance after glucocorticoid treatmentObjective: Severe hepatitis B(SHB) is the most dreaded type of hepatitis B because of its fast progression and low eradication rate. Therefore, treatment in the early stage of SHB is critical. Our study focused on Th17, Treg and Th17/Treg alterations in the early stage of SHB and the effect of glucocorticoids on these immune cells. Methods: The study included 20 patients in the early stage of SHB and 11 healthy controls. The patients met the following inclusion criteria: extreme fatigue with severe gastrointestinal symptoms such as anorexia, vomiting, and bloating; elevated jaundice level(51 μmol/L ≤ total bilirubin [TBil] ≤ 171 μmol/L) and daily increase > 17 μmol/L; bleeding tendency with 40% ≤ plasma thromboplastin antecedent [PTA] ≤ 50%(or 1.5 ≤ international normalized ratio [INR] ≤ 1.6); and positive markers for HBV. Fasting peripheral venous blood(2 m L) was drawn from patients and healthy controls in the morning. The collected blood was put into test tubes with sodium heparin for the detection of specific immune cells(before, during, and after treatment) by intracellular cytokine identification using in vitro stimulation and fixation/permeabilization followed by flow cytometry. Results: All patients had elevated Th17 levels, decreased Treg levels, and significant Th17/Treg ratios. After glucocorticoid treatment, 16 patients showed improvement with significant decreases in Th17 levels, increases in Treg, and rebalanced Th17/Treg ratios. The four patients who showed no improvement had increases in both Th17 and Treg levels and an even higher Th17/Treg ratio than before. Conclusions: Th17/Treg dysregulation was present in all patients in the early stage of SHB. If glucocorticoid treatment results in decreased Th17 levels, increased Treg levels, and, therefore, rebalanced Th17/Treg ratios, then it is very likely that the condition can be eradicated. If Th17/Treg rebalance does not occur after glucocorticoid treatment, SHB will likely progress and eventually cause death.