| Toll like receptors(TLRs) that are regarded as pattern recognition receptors(PAMPs) in innate immune system can specifically recognize conserved pathogen associated molecular patterns of pathogenic microorganisms and regulate immune responses by activating downstream signaling pathways. TLR4 is widely expressed in liver parenchymal and non-parenchymal cells and it also plays a vital role in physiological and pathological processes in liver through identifying lipopolysaccharide(LPS) which is a major pathogenic component of Gram-negative bacteria. Prior studies have confirmed that the LPS/TLR4 signaling pathway is involved in the occurrence and development of a variety of liver diseases. The previous research investigations on the relationship between TLR4 signaling pathway and liver injury were mostly conducted in mammals, while only a few reports described the dynamic changes in TLR4 mediated liver injury in avian species. Therefore, in the current study, one-day-old Cobb strain chickens were used for the identification of molecular events involved during LPS and Salmonella Typhimurium stimulation and for clarification of the relationship between TLR4 signaling pathway and liver injury at different time points. In the current study, different staining methods including HE, chromotrope 2R, and immunohistochemical staining along with Q-PCR were used to study the changes in liver tissue structure, hepatic cell apoptosis and dynamic expression of inflammatory cytokines, meanwhile the changes in the expression of TLR4 and downstream signaling molecules were also detected after LPS and Salmonella Typhimurium stimulation. The main research contents and results are as follows: 1. Effects of LPS or Salmonella Typhimurium stimulus on the histological structure and ALT activity of chicken liverThis research was performed to explore the alterations in chicken liver structure and alanine aminotransferase(ALT) activity through intraperitoneal injections of LPS and Salmonella Typhimurium in 1-day-old Cobb broilers. Results: after intraperitoneal injection of LPS at 6h, 12 h and 24 h, liver showed severe fatty degeneration and necrotic symptoms, the number of heterophil granulocyte in liver sinus was significantly increased, and at 72 h and 120 h it mainly manifested a wide range of inflammatory cell infiltration. Compared with the control group, heterophil granulocytes also significantly increased at each time after Salmonella Typhimurium stimulation, but no obvious pathological changes were observed at 2h and 6h, however, with the passage of time, liver exhibited steatosis, ballooning degeneration and congestion at 24 h and 36 h, and less amount of inflammatory cell infiltration around the portal area were observed at 72 h. ALT activity was significantly higher than that of control group after LPS or Salmonella Typhimurium stimulation and it first increased and then decreased in time dependent fashion, which attained its peak at 12 h and 24 h after LPS and Salmonella Typhimurium stimulations respectively. These results indicate that LPS or Salmonella Typhimurium can induce acute injury in chicken liver.2. Effects of LPS or Salmonella Typhimurium stimulus on the expression of inflammatory cytokines in chicken liverIn order to study the expression changes of inflammatory factors in liver after LPS or Salmonella Typhimurium stimulation, Q-PCR was used to detect the expression changes in TNF-α, IL-1β and TGF-β. Results: the expression of TNF-α, IL-1β and TGF-β increased first and then decreased after LPS stimulation, and showed an extensive increase at 2h, then began to decline at 6h. However, their expression level was higher than that of the control group at each time after LPS stimulation. Compared with TNF-α and IL-1β, the growth rate of TGF-β was lower. The changes in the expression pattern of inflammatory factors were similar in both LPS and Salmonella Typhimurium groups: The anti-inflammatory and pro-inflammatory cytokine expressions reached their peak at 24 h, although their levels were higher than that of the control group, but the changes were not as drastic as in LPS group. These results show that LPS or Salmonella Typhimurium stimulation can cause an imbalance in the expression of anti-inflammatory and pro-inflammatory cytokines, which lead to liver damage. In the present study, the changes in the expression of inflammatory factors caused by Salmonella Typhimurium stimulation were weaker than that of LPS, and these findings were also consistent with liver organizational as well as structural changes after the LPS or Salmonella Typhimurium stimulation. 3. Effects of LPS or Salmonella Typhimurium stimulus on the hepatocyte apoptosis and proliferation in chicken liverIn order to study the influences of LPS or Salmonella Typhimurium stimulation on hepatocyte apoptosis and proliferation, immunohistochemical staining method was used to investigate the changes in the expression of ssDNA and proliferating cell nuclear antigen(PCNA); caspase-3 activity assay kit was used to detect the changes of caspase-3 activity in chicken liver; Q-PCR was performed to study the alterations in hepatic caspase-3, caspase-8 and PCNA expressions. Results: apoptosis mainly happened at 2h, 6h and 12 h after LPS stimulation, and was significantly inhibited at 24 h after stimulation. This finding was consistent with the results of caspase-3 activity assay: activity of caspase-3 showed first increased and then decreased trend reaching at a peak level after 12 h. Moreover, the activity of caspase-3 in 2h, 6h and 12 h groups was extremely significant than that of control groups after LPS stimulation, but was significantly lower than that of control groups at 24 h after stimulation. The m RNA expression of both caspase-3 and caspase-8 reached the peak at 2h after LPS stimulation however it was lower than that of the control group at 12 h. While the mRNA expression of caspase-3 and caspase-8 attained the peak level at 24 h after the stimulation of Salmonella Typhimurium, but the caspase-3 activity was not significantly increased after Salmonella Typhimurium stimulation, and was significantly lower than that of the control group at 12 h and 24 h. The expression of PCNA was significantly decreased after LPS or Salmonella Typhimurium stimulation however in the later stages its expression was slowly increased to the level of the control group. These results suggest that LPS stimulation can promote the apoptosis and inhibit the proliferation of hepatic cells which lead to liver injury, furthermore, the hepatocyte apoptosis was mainly mediated by activated caspase-3; while Salmonella Typhimurium stimulation didn’t cause hepatocyte apoptosis, but mainly led to liver damage by inhibiting the proliferation of liver cells. 4. Effects of LPS or Salmonella Typhimurium stimulus on the expression of TLR4 signaling pathway and its related molecules in chicken liverIn order to study the changes in molecular expression of TLR4 signaling pathway after LPS or Salmonella Typhimurium stimulation, immunohistochemical staining was used to study the expression changes in TLR4. Q-PCR was performed to detect the expression changes in TLR4, NF-κB and MyD88, both NF-κB and MyD88 are known as downstream molecules of TLR4 signaling pathway. Results: the expression pattern of TLR4 varied at different time points after LPS stimulation, that showed an extensive increase at 6h, but it was lower than that of control group at 24 h and 36 h, again it was significantly higher than that of control group at 72 h and 120 h. The expression of TLR4 after Salmonella Typhimurium stimulation was increased first and then decreased, reached the peak at 24 h, and began to decrease at 36 h, 120 h and was even lower than that of the control group at these time points. The expression of NF-κB were higher than the control group after LPS stimulation, and reached its peak at 2h. The expression of NF-κB after Salmonella Typhimurium stimulation peaked at 24 h, and decreased below the control group at 120 h. The expression of MyD88 in liver after LPS stimulation showed an extensive growth at 2h, began to decline at 6h, and was significantly lower than that of the control group at 36 h. The expression of MyD88 in liver after Salmonella Typhimurium stimulate peaked at 24 h, 36 h began to decrease at 120 h and was significantly lower than that of the control group. These results indicate that LPS or Salmonella Typhimurium can lead to liver damage by inducing the expression of inflammatory cytokines and liver cell apoptosis through the TLR4 signaling pathway. |
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