| [Background]Hepatitis B virus associated glomerulonephritis (HBV-GN) is one of the main extrahepatic manifestation of HBV infection, while, renal tissue damage caused by immune complex is a recognized the pathogenesis of HBV-GN. Today immune electron microscopy for HBV-GN kidney tissue ultrastructural morphology observed the presence of HBV in renal inherent cells, and PT-PCR detection technology directly detected HBV-DNA replication in HBV-GN renal tissues, suggests that in addition to HBV antigens-antibody complex in glomerular deposition and pathogenesis, should also consider the kidney injury mechanisms caused by HBV directly infection may also involve in the pathogenesis. Accordingly, some scholars put forward the point of view that:HBV can directly infect, replicate and damage in renal tissue. Though the above research and evidence to support this view, it is only morphological observation or analysis inference, rare of the relevant research to clarify the virus directly damage mechanism at the molecular level, therefore still need further discussion, to reveal the molecular pathways of HBV virus itself in disease.The DNA in the cytoplasm is a powerful motivating factor of the body’s immune response, the downstream signal channel involved in the process of the excitation (or in the trigger process) has been widely research and reveal, but the identification(or recognition) of body to the exogenous DNA in cytoplasm and trigger mechanism is poorly understood.In recent years, immunological basis study found that:PYHIN protein member--IFI16can feel cytoplasmic and nuclear dsDNA, initiate a natural immune response, induce IFN-β and inflammatory cytokines; The other members of the family-AIM2can combine with exogenous dsDNA transfected into the cytoplasm, promote the activation of Caspase-1, form of inflammatory complex.Different studies have demonstrated that after cells infected, the exogenous dsDNA in the cells would activated AIM2and continued to express, become an important inflammatory reactants and triggered a series of intracellular reaction, leading to inflammatory factor IL-1β, IL-18secretion and release, such as play inflammatory and apoptosis, and other important biological activities.The above-mentioned study content are reported in recent years, but are limited to immunology and tumor etiology rese arch, the research reports in infectious disease are mainly concentrated in bacterial infections, a few studies on HBV with AIM2mainly for basic research. This research set two PYHIN family protein-IFI16and AIM2as the research target, the feature of who can be perceived a series of inflammatory after recognition to the dsDNA in the cells is applied to study the pathogenesis of HBV-GN, discuss whether HBV DNA in renal tissues can active IFI16and AIM2in cells, and the specific pathogenic mechanism in the clinical disease--HBV-GN after their activation.[Methods]Histology and cytology experiments in vitro were applied as two route to study. Applied kidney puncture biopsy pathology technique (including HE staining and special staining), immunofluorescence staining, immunohistochemical staining and electron microscope technology, combined with45cases of HBV-GN which were clinical clear diagnosis and found HBV virus particles existing in renal tissues by screen, and applied immunohistochemical staining to detect the expression of IFI16, IFN-β, AIM2, Caspase-1, IL-1β, IL-18in tissue, in order to be clear and definite the correlation of HBV infection in the kidney tissues between the expression of IFI16and AIM2, in order to be clear and definite the correlation between IFI16activation and IFN-β expression, and in order to be clear and definite the correlation between AIM2activation and expression of Caspase-1and its downstream factor IL-1β and IL-18.In vitro cultured kidney inherent cells (including HRGEC, HRMC and HRPC) were transfected by pcDNA3.0-1.1HBV, then stained with fluorescence double labeling and detected using PT-PCR respectively, positioned and quantitative determinate six indicators level (including IFI16, AIM2, IFN-β, Caspase-1, IL-1β and IL-18) in glomerular inherent cell cytoplasm which was infected by HBV, compared with the non-transfection group, analyzed the correlation; localized observation and validated the relationship between HBV and IFI16and AIM2activation using fluorescence double labeling technic; quantitative detected intracellular expression (including IFI16, IFN-β, AIM2, Caspase-1,1L-1β and IL-18) of HREC HRMC and HRPC which were transfected by HBV, in order to discuss the relationship between IFI16activation and IFN-β expression, between AIM2activation and expression of Caspase-1, the formation of inflammatory complex and the correlation with the secretion and release of the downstream inflammatory cytokines (IL-1β and IL-18) after the HBV-DNA trigger the activation and expression of IFI16and AIM2.In cell experiments, constructed cryptogenic siRNA, set up the HBV DNA-IFI16/AIM2siRNA co-transfection group, repeated measurement the content of IFI16, AIM2, Caspase-1and downstream factor within cells under the condition of HBV infection, analyzed the correlation between two targets. Validated in two-way in the same experiment route, in order to be clear and definite that HBV-DNA activated IFI16and AIM2is the key of trigger to the virus directly damage, and inflammatory complex formation and lead to inflammatory lesions play an important role in the pathogeny of HBV-GN. Two routes supported each other. Meanwhile, observed and analyzed if there is relativity between the activity of HBV and inflammatory composite of IFI16and AIM2and degree of renal tissue damage, and the type of morphological change.[Results]Electron microscopy testing found that19.07%of HBV-GN patient’s kidney tissues have HBV virus particles, which can be used as a supplementary means for diagnosis, revised light microscopy diagnosis, reduce missed diagnosis or misdiagnosis for those cases that HBsAg, HBcAg and HBeAg are negative by histologic examination and cannot be diagnosed HBV-GN cases according to the diagnostic criteria.HBV-GN clinical manifestations are complicated. The renal damage which most commonly occurs in clinical have the manifestations which are proteinuria, followed by hematuria merge proteinuria or nephrotic syndrome. They have varied morphological performance, in which, the membranous nephropathy, membranous nephropathy associated with atypical changes and membrane proliferative glomerulonephritis are more common. The average age of onset are36.512.5years old and the incidence of men are more than women.The expression of IFI16and AIM2in HBV-GN patients’ renal tissue is significantly higher than the control group, both of them are closely associated with HBV infection. The intensity of IFI16expression of HBV-GN group is positively related to the intensity of IFN-β expression and the same as AIM2vs. Caspase-1, IL-1β and IL-18vs. Caspase-1.The detection of mRNA and protein level of IFI16, IFN-β, AIM2, Caspase-1, IL-1β and IL-18in three-line cells indicate that:compared with the control group, mRNA level and protein expression in six indicators of pcDNA3-3HBV transfection group significantly increased, in which, the intensity of IFI16expression is positively related to the intensity of IFN-β expression and the intensity of AIM2expression is closely associated with the intensity of the expression of Caspase-1, IL-1β and IL-18. To compare the siRNA and HBV cotransfection group with the pure HBV transfection group, all measurements are reduced. The results indicate that, blocking IFI16can make the quantity of IFN-P expression decreased obviously, in the same way, after blocking AIM2, the quantity of caspase-1, IL-1β and IL-18expression decreased obviously, too. RT-PCR and Western blot have the same test results.[Conclusion]In summary, we arrive at the following review. In HBV-GN, two proteins of PYHIN family members--IFI16and AIM2can perceive intracellular HBV DNA, both of them can activate innate immunity through two pathways after expression infection leading to the activation, and participate in the occurrence of the HBV-GN, which including two pathogenesis:1. Inflammatory factor IFN-β is induced after IFI16been activated;2. Activated AIM2combine with Caspase-1to form inflammatory complex and release the inflammatory factors IL-1β and IL-18. We confirm that, in addition to the classic immune complex damage ways, HBV can infect directly in renal tissue, result in tissue damage. |
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