The Effect And Mechanism Of Heat Shock Protein 47 In Hepatic Fibrosis Of Schistosomiasis Japonica

[Background and aims]Hepatic fibrosis is a self defense and reparing response to chronic liver injury in an intermediate stage during the development of liver disease. Chronic liver diseases such as viral hepatitis, schistosomiasis liver disease and alcoholic hepatitis undertake this common process, which eventually lead to liver failure. Delaying and preventing the development of liver fibrosis is the key for prevention and treatment of disease progression. Around the world, there are about 200 million people threatened by schistosomiasis. There are still new Japanese schistosomiasis case reports in 12 provinces in China and liver cirrhosis caused by Schistosoma japonicum. Researchers found that the liver fibrosis is a dynamic, reversible process caused by imbalanced proliferation and degradation of extracellular matrix. Many studies indicated that persistent activation of hepatic stellate cells is the key link in the development of liver fibrosis.HSCs are in a resting state under physiological conditions. When the liver is subject to various causes of stimulations (such as schistosome eggs induced typeⅣhypersensitivity), HSC phenotype and function changes, such as the loss of lipid droplets and vitamin A, overexpression of some proteins, such asⅠ-Smooth muscle actin (α-SMA), synaptophysin (syn), heat shock protein 47 (HSP47), and several cytokines, such as transforming growth factor-β1 (TGF-betal) and connective tissue growth factor (CTGF) were released and amplificated through the cascade of cytokines to promote their self-activation. After which ECM with a large number of collagen fibers (ⅠandⅢcollagen) was synthesized and secreted. Enzymes Regulating ECM degradation include matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase (TIMPs). MMPs These enzymes are synthesized and secreted by fibroblasts, smooth muscle cells, endothelial cells, macrophages and neutrophils They are a endogenous proteolytic enzyme family, involving in extracellular matrix degradation of a class of zinc, and this effect is calcium dependent. These enzymes are also physiological regulators of extracellular matrix and play an important roles in the process of degradation and destruction. TIMPs are a group of multifunctional factor family inhibiting the activity of MMPs. Currently, four family members of TIMPs have been found and were named as TIMP1, TIMP2 TIMP3 and TIMP4. MMP and TIMP form MMP-of TIMP complex in a 1:1 ratio, thus blocking the MMP and collagen substrate binding and inhibiting the activity of MMPs, resulting in collagen degradation. Imbalance of collagen degradation eventually leads to the aggregation of ECM and causes hepatic fibrosis. Currently, there is another hypothesis suggesting that the fibrinolytic system has a dual role in the generation and progress of liver fibrosis. On one hand, it can degrade perisinusoidal ECM and destruct the normal contact between the cells and the level to promote the proliferation and migration of HSCs (I do not understand this sentence). On the other hand, the activation of TGF-(31 increases the synthesis of PAI-1 and TIMPs, thus promoting the synthesis and deposition of ECM and inhibiting plasmin generation and matrix degradation.HSP47 is a stress protein synthesized in the body upon the stimulation of stress. Most recently, according to researches on HSP47's role in the scarring of skin and eyes, HSP47 can specifically bind to varies types of collagen and procollagen peptides and assist to correct collagen folding to form a unique tertiary structure. Thus it is a necessary partner in the process of synthesis and secretion of procollagen. HSP47 is a heat shock protein with a molecular weight of 47KD. It is mainly involved in collagen synthesis in the endoplasmic reticulum of cells (such as hepatic stellate cells) and its role in fibrosis formation is still at the exploratory stage. It has been found that HSP47 upregulation is consistent with collagen synthesis and fibrosis progression. Inhibition of collagen synthesis reduces collagen synthesis and fibrosis. However HSP47's roles in the development of hepatic fibrosis due to schistosomiasis, especially its effect on HSC activation, are seldomly studied.Genetic level intervention of a "bad" gene expression not only can better elucidate the important role of the gene in the development of the disease but also explore new insights for the clinical treatment of liver fibrosis. Some studies found that efficient expression of plasmid-DNA in mouse liver can be achieved by tail vein hydrodynamic injection. By doing so allows a large volume of naked plasmid-DNA solution quickly get into the mouse circulation, resulting in a sharp increase in the blood volume and heart overload., Upon this situation the blood accumulated in the hepatic sinusoid cannot flow back and the residence time of plasmid-DNA in the liver sinusoidal extends. Additionally, most of the drugs first metabolize in the liver, so the plasmid-DNA in liver tissue can be uptaken. Now this technology has been widely applied in the study of genetic intervention for controlling liver diseases.In summary, HSP47 is a specific molecular chaperone of collagen synthesis(I do not understand what you want to say here), which is upregulated during the course of liver fibrosis and it in turn promotes liver fibrosis. However its expression profile and contribution in the development of liver fibrosis due to schistosomiasis japonica and subsequently related mechanisms are not fully understood. AimThis study was aimed to investigate the expression profile of HSP47 and its contribution in the development of liver fibrosis due to schistosomiasis japonicaMethodLiver biopsy specimens from patients (48 cases) with liver fibrosis of schistosomiasis and the clinical data were collected from Tongji Hospital outpatient and inpatient department between 2008-2011. HSP47, cytokines (TGF-β1 in CTGF) and typeⅠcollagen expression in the liver were detected by immunohistochemistry and real-time PCR. In addition, HE and Masson staining were performed to observe pathological changes and the deposition of collagen fibers.liver fibrosis mouse model induced by schistosomiasis was established. Female Balb/c mice of 6 weeks old were infected with schistosome cercariae through abdominal skin. At 6 or 12 weeks post infection, the livers of mice were collected and liver tissue paraffin sections were produced for HE and Masson staining. Immunohistochemistry and real-time PCR reaction were used to detect HSP47, cytokine (TGF-β1) and typeⅠcollagen expression.Result1. Compared to the normal control, there were a large number of fibrotic tissues proliferating and depositing in the portal and hepatic lobule areas at stage S2 without formation of interlobular septal. In phase S4, the hepatic lobule structures were gradually destroyed and fiber intervals were observed between the central vein area and the portal area.2. Mason staining results indicated that liver fibrosis tissue and collagen in the liver interstitial area significantly increased compared to the normal control.3. Immunohistochemical results showed that the expression of HSP47 in liver fibrosis group (S2-S4) significantly increased in the portal area and interstitial area fibrosis compared to the control; the expression of stellate cell activation marker (alpha-SMA) increased in the portal and the interstitial areas; TGF-β1 expression increased significantly mainly in inflammatory infiltration and fibrosis areas.4. Real-time PCR results indicated that in liver fibrosis tissues HSP47, TGF-betal, CTGF collagenⅠand collagenⅢmRNA levels significantly increased compared to the normal control.ConclusionHSP47 and TGF-betal, CTGF were upregulated within fibrotic tissues in the liver in patients with chronic schistosomiasis japonica infection as well as mice post schistosomiasis japonica infection. Further more the HSP47 and collagen co-expressed was observed in the same areas in both patients and mice. These results suggested HSP47 expression is closely related to collagen synthesis and in turnmay play a role in the development of liver fibrosis in chronic schistosomiasis japonica infection. AimConstruct a shRNA against liver fibrosis key gene-HSP47. Test whether it interfered with the HSP47 gene expression in NIH/3T3 cell lines in vitro and observe the influence on the cell function.Method1. Design HSP47-shRNA template at the downstream of U6 promoter. Amplify U6 promoter by PCR and attach template double-stranded DNA to the downstream of U6 promoter. After T vector and the pGCsi-U6/Neo/GFP transition, load U6 promoter and HSP47 shRNA template at its downstream to get HSP47-shRNA.2. Transfect HSP47-shRNA and non-interference vector as a control. Cells were collected at 12,24 and 48 hours respectively. Assess HSP47 expression at the mRNA and protein levels by real-time PCR and western-blot and observe collagen secreted by the cells.Result1) Get DNA through PCR amplification.The DNA fragments were proved by BamHⅠand HindⅢdouble restriction enzymes digestion.Verify expected DNA sequences by sequencing double-stranded DNAs of U6 promoter and HSP47-shRNA template. Observed significantly more cells expressing green fluorescent protein compared to the control group.2) Measured by RT-PCR and western blot, HSP47 mRNA and protein expression significantly decreased in the intervention group.Detected by RT-PCR, TGF-β1 expression in the intervention group decreased significantly.3) Checked by PCR and ELISA, collagenⅠandⅡsynthesis significantly reduced in the intervention group. ConclusionHSP4-shRNA interference plasmid was successfully constructed. Our primary data confirmed it effectively interfered the expression of HSP47, laying a foundation for further experiments in vivo. AimObserve the expression of HSP47-shRNA in mouse liver to further explore the regulation of HSP47-shRNA on the network of cytokines and eventually elucidate HSP47's role in progression of mouse liver fibrosis.Method1) Establish a mouse model with schistosomiasis. Detect target gene expression in the liver in vivo after tail vein hydrodynamic injection of HSP47 -shRNA in mouse.2) Mice survival rate was observed after tail vein hydrodynamic injection. Liver pathology and serum biochemical changes were also observed.3) HSP47, collagen and cytokines (TGF-β1, CTGF, MMP-9, TIMP-1, IL-13 and IL-17) were detected by real-time PCR, immunohistochemistry, ELISA and western blot.Result1) In mice treated with different concentrations of HSP47-shRNA interference, survival rates in high, median or low dose group improved to 33.33%,25% and 25% respectively 14 weeks after infection,compared to the control of 16.67%.2) Compared to the control, HE staining results showed the significantly narrowed (specific values) schistosome egg granuloma and obviously reduced liver fibrosis in the interference group. Masson staining indicated significantly reduced hepatic collagen deposition in the interference group.3) After interference of HSP47, cytokines related to liver granuloma reaction and liver fibrosis (TGF-β1,CTGF,IL-13,IL-17,MMP-9,TIMP-1 and PAI-1) changed differently. The expression of stellate cell activation marker(α-SMA) and collagenⅠ/Ⅲsignificantly decreased. ConclusionInterference with the expression of HSP47 significantly improved the liver fibrosis. HSP47 not only influences collagen expression but also has an impact on the cytokine regulatory network related to liver fibrosis. Further studies are needed to uncover its detailed mechanism.