| Liver disease is a major health and economic burden worldwide.With the increasing incidence of liver disease,it has become one of the major causes of death and disease in the world.In various chronic liver diseases,with the progress of liver disease,hepatocytes gradually lose their strong regenerative ability and undergo cell aging or death,which will lead to fibrosis,cirrhosis,and even liver cancer.When the liver disease progresses to the end stage,the only effective treatment is liver transplantation.However,immunosuppressive agents need to be taken for life after transplantation.At the same time,the lack of donor organs means that patients may die before waiting for the appropriate organ transplantation.In clinical practice,liver regeneration is the prerequisite to ensure the survival and good prognosis of patients with liver injury caused by partial hepatectomy and partial liver transplantation.When the liver cannot regenerate effectively after partial hepatectomy or partial liver transplantation,it will cause the small-for-size syndrome that seriously threatens the life of patients.In patients with acute and chronic liver injury caused by drugs,hepatotoxic substances,and other factors,the effective proliferation of hepatocytes is also an important guarantee for a good prognosis of patients.Therefore,it is of great clinical significance to establish effective means to initiate liver regeneration or repair the damaged regeneration ability of hepatocytes.It is well known that the liver has the excellent regenerative ability,which was first recorded in the Greek myth-Prometheus stole kindling.The rodent model of two-thirds hepatectomy showed this powerful regeneration ability for the first time.The hepatocytes in the normal adult liver are in the G0 phase and rarely undergo cell division.However,after partial hepatectomy,about 95% of the hepatocytes quickly re-enter the cell cycle,and the remaining liver quickly returns to its original size in about one week.Liver regeneration is a multi-step,multi-factor,and multi-signaling regulated process.Due to the complexity of the process of liver regeneration,many regulatory mechanisms still need to be further explored.To sum up,clarifying the regulatory mechanism of the initiation and termination of liver regeneration is of great significance for clarifying the etiology,pathogenesis,and clinical treatment of many liver diseases(such as metabolic liver disease,cirrhosis,liver cancer,etc.).Therefore,we try to precisely intervene liver homeostasis through small molecule combination to achieve accurate and effective regulation of liver regeneration in situ.This study mainly includes four parts.The first part is the chemical screening of active molecules to promote the proliferation of hepatocytes in situ.Based on our previous research and the mechanism of liver regeneration after injury,the TGF β receptor inhibitor,lysophosphatidic acid,the p38 MAPKs inhibitor,the prostaglandin E2 derivative,and the Toll-like receptor agonist were selected as the basal molecules to screen the active molecules and combinations that promote cell proliferation in situ in the liver.First,the TGF β Receptor inhibitor was selected as the basal molecule to screen the active molecular combination of promoting liver cell proliferation in situ.The TGF β receptor inhibitor was selected as the basal molecule combined with two other important signal pathways Wnt and HGF/c-Met in the process of liver regeneration to try to start cell proliferation in the liver.The results showed that the combined regulation of TGF β,Wnt,and HGF/c-Met signal pathways could not effectively induce the proliferation of mouse liver cells in situ.Given that liver regeneration is a complex process involving multiple signal pathways,based on the above combination we added the small molecular compound Resiquimod,the agonist of TLR7/TLR8,and chenodeoxycholic acid,the agonist of farnesoid X receptor.The results showed that the addition of the agonist of TLR7/TLR8 and the agonist of farnesoid X receptor could not effectively promote the proliferation of mouse liver cells in situ.Because of the importance and diversity of Toll-like receptors in the initiation of liver regeneration,We replace the agonists of TLR7/TLR8 with another TLR agonist S.The results showed that the addition of TLR agonist significantly increased the proportion of Ki67-positive cells in mouse liver(6.27% ± 0.83%).We speculated that the TLR agonist might play an important role in liver regeneration.Next,the TGF β receptor inhibitor was selected as the basal molecule,which was combined with the Toll-like receptor agonist(LS combination),the nuclear receptor agonist(LT and LW combination),and the Nrf2 agonist(LC combination)to explore its effect on the proliferation of liver cells in situ.The results showed that the four groups of combinations increased the proportion of Ki67 positive cells in situ in mouse liver at different degrees,and the positive proportion of LS combination and LT combination was18.83% ± 0.92% and 11.82% ± 1.82% respectively.The next step is to screen the effect of lysophosphatidic acid and its combination on liver cell proliferation in situ.Through screening,it was found that the proportion of Ki67-positive cells in the liver of mice treated with lysophosphatidic acid(LPA)alone was only 4.39% ± 0.95%.The addition of TGF βreceptor inhibitor and GSK3 inhibitor decreased the proportion of Ki67-positive cells.The next step is to explore the role of a p38 MAPKs inhibitor and its combination in promoting hepatocyte proliferation in situ.The results showed that the treatment of p38 MAPKs inhibitor alone had no significant effect on the proportion of Ki67 positive cells in situ in mouse liver.When it was combined with TGF β receptor inhibitor and GSK3 inhibitor respectively,it could increase the proportion of Ki67 positive cells in situ in mouse liver,but the proportion of Ki67 positive cells was only about 2%.Next,we explored the effect of prostaglandin E2 derivative and its combination on hepatocyte proliferation in situ.The screening results showed that the low-dose group treated with the derivative of prostaglandin E2 could start the proliferation process of mouse liver cells in situ,and avoid the liver injury and mouse death caused by the high-dose group.The combination of prostaglandin E2 and the TGF β receptor inhibitor could not effectively increase the proportion of proliferating cells in situ in mouse liver,while the combination of prostaglandin E2 and GSK3 inhibitor could significantly improve the proliferation ability of mouse liver cells in situ,which was higher than the low-dose group treated alone.Finally,based on Toll-like receptor agonists,active molecules that promote hepatocyte proliferation in situ were screened.Based on the preliminary screening results,we speculate that a Toll-like receptor agonist plays an important role in the initiation of liver cell proliferation in situ.We select the CR an agonist of Toll-like receptor to explore the effect of small molecule compound CR on the initiation of liver cell proliferation in situ.The results showed that the proportion of proliferative cells in the liver of mice treated with Toll-like receptor agonist CR was 12.66% ± 2.94%,significantly higher than that in the control group.Based on the above screening process,we finally choose the combination of TGF β receptor inhibitor and Toll-like receptor agonist(LS),the combination of TGF β receptor inhibitor and nuclear receptor agonists(LT),the combination of the derivative of PGE2 and GSK3 inhibitors(dm C2)and toll-like receptor agonist CR as the molecules for further research.The second part is to explore the role of the active small molecule compound and three combinations in the model of nonalcoholic fatty liver disease.Studies have shown that fatty liver can inhibit normal regeneration processes,so we attempt to explore the efficacy of active small molecule compounds and combinations in nonalcoholic fatty liver disease models.The results showed that the combination of LS and LT partially reversed fatty degeneration in the nonalcoholic fatty liver mice model,but caused the death of model mice.In the nonalcoholic fatty liver model mice,the treatment of small molecular compound combination dm C2 or CR significantly improved the liver tissue structure,reduced the extension of fatty degeneration,collagen deposition,and hepatic stellate cell activation,promoted the proliferation of hepatocytes,and significantly increased the liver/body weight ratio of the model mice.At the same time,the liver function index and blood lipid index of mice were significantly improved.Therefore,we choose the small molecular compound CR and the combination of dm C2 for further investigation in this study.The third part is the evaluation of the structure and function of the liver and the toxicity of small molecular compounds.It is well known that liver injury can initiate liver regeneration.Therefore,we evaluated the liver structure and serum liver function indicators of mice treated with the molecule and combination,excluding the possibility of liver injury,which is caused by the toxic side effects of small molecule compounds,leading to liver regeneration The results showed that the liver lobule structure of mice was intact and clear,and there was no obvious lesion.The serum liver function indicators were not beyond the normal range.Next,we evaluated the expression of liver functional molecules under the treatment of the molecule and the combination,and the results showed that there was no abnormal change in liver functional molecules.Co-staining with Ki67 immunofluorescence showed that most of the proliferating cells were hepatocytes,while co-staining with liver metabolic enzyme CYP2E1 and Ki67 immunofluorescence suggested that proliferative hepatocytes are mainly distributed around the central vein in the hepatic lobular zone II.Next,the liver structure was evaluated under the conditions of the molecule and the combination treatment.Phalloidin staining showed normal f-actin network.DPP4 staining revealed clear bile canaliculus stucture.Ki67 co-stained with the hepatic progenitor cell marker SOX9 and AFP showed that the compound treatment initiated the liver regeneration without activating hepatic progenitor cells.Finally,the localization of proliferating hepatocytes in hepatic lobules under the treatment was investigated.Ki67 and β-Catenin immunofluorescence co-staining showed that Ki67 positive proliferative hepatocytes were mainly distributed in zone II between the central and portal veins.Further,the zone I marker E-CAD,the zone III marker GS,and the cell proliferation index Ki67 immunofluorescence co-staining showed that the proliferating hepatocytes mainly located in the zone II.The fourth part is the preliminary study of the molecular mechanism of the chemcial compound induced hepatocyte proliferation.It is well known that the Hippo signal pathway plays an important role in liver regeneration after injury.However,YAP depletion in hepatocytes did not significantly compromise the proliferation of hepatocytes after the compound treatment.Next,we analyzed the the transcriptome expression profile of hepatocytes after compound treatment.The transcriptome data showed that the down-regulated genes accounted for 78.3% of the differential genes in the experimental group treated with the combination of dm C2.The enrichment analysis data showed that the negative regulatory pathways of cell proliferation in the control group were significantly enriched,suggesting that the combination of dm C2 might start liver regeneration mainly by down-regulating the negative regulatory signal pathway related to cell proliferation.Different from the the treatment of dm C2,90.59% differential genes in the experimental group of small molecular compound CR were up-regulated genes.Further,the enrichment analysis data showed that the signal pathways related to cell proliferation were significantly enriched in after small molecular compound CR treatment,suggesting that small molecular compound CR might start liver regeneration mainly by up-regulating the signal pathway related to cell proliferation.As CR having a defined target,we further explored whether the small molecule compound S,which has the same target as CR,can also start liver regeneration.The results showed that the compound S alone can also significantly increase the hepatocyte proliferation in situ in the liver of mice,confirminging that the target has an important role in the proliferation of liver cells in situ in mice.In addition,for the small molecule compound CR,we explored the role of its downstream molecule TNF-αin liver regeneration.The results showed that the treatment of TNF-αinhibitor adalimumab did not significantly reduce the liver weight ratio and the number of hepatocytes proliferating in situ in the liver of the experimental group.In this study,wild-type mice were used to carry out the chemical screening of active molecules that promote the proliferation of liver cells in situ.After the screening,there groups of small molecular combinations and a small molecular compound that can significantly initiate liver regeneration in situ were obtained.After optimizing the drug delivery and dosage,we explored the role of the dm C2 and CR in the model of nonalcoholic fatty liver disease.We found that the dm C2 and CR can significantly improve the liver tissue structure and reverse steatosis of model mice,but the combinations of LS and LT led to a certain proportion of mouse deaths.Routes and dosages of drug administration need to be further optimized.The combinations of dm C2 and the small molecular compound CR did not cause the death of the model mice.Further studies showed that dm C2 and CR reduced the proportion of collagen deposition and hepatic stellate cell activation,promoted the proliferation of cells in the liver,significantly increased the liver/body weight ratio of the model mice,and significantly improved the liver function index and blood lipid index of the mice.Next,we evaluated the structure and function of the liver after compound treatment,the location of proliferating hepatocytes in the liver lobules,as well as the toxicity of these small molecular compounds.Finally,we made a preliminary study on the molecular mechanism of the active molecule and the combination to promote hepatocyte proliferation,and we will continue to further explore their mechanism.Through the research of this project,we have established effective chemical intervention measures to promote liver regeneration,provided new insight of liver regeneration,which could be useful for the treatment of liver injury and other related diseases. |
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