Senescence suppressed proliferation of host hepatocytes is precondition for liver repopulation.

In the fumarylacetoacetate hydrolase deficient (Fah-/-) mouse, massive liver repopulation can be easily obtained after transplanted hepatocytes. Understanding the mechanisms of complete liver repopulation in Fah-/- mice will be useful for future clinical application. Here, we found that the endogenous hepatocytes in liver of Fah-/- mice undertook senescence during the time of tyrosinemia symptoms. Increase of senescent hepatocytes in Fah-/- mice provided proliferative advantage to the transplanted hepatocytes. Importantly, senescent hepatocytes upregulated the expression of extracellular matrix enzyme, contributing to degradation of extracellular matrix components and weakness of cell adhesion and connection. The liver exhibiting a loose architecture provided the space for the engraftment and expansion of transplanted hepatocytes. These findings underscore the underlying mechanisms of completed liver repopulation in Fah-/- mice. Senescence followed by loose hepatic parenchyma is a preconditioning for liver repopulation, which would be a promising strategy to achieve therapeutic liver repopulation in clinical settings.

Reversal of hepatocyte senescence after continuous in vivo cell proliferation.

UNLABELLED: A better understanding of hepatocyte senescence could be used to treat age-dependent disease processes of the liver. Whether continuously proliferating hepatocytes could avoid or reverse senescence has not yet been fully elucidated. We confirmed that the livers of aged mice accumulated senescent and polyploid hepatocytes, which is associated with accumulation of DNA damage and activation of p53-p21 and p16(ink4a)-pRB pathways. Induction of multiple rounds continuous cell division is hard to apply in any animal model. Taking advantage of serial hepatocyte transplantation assays in the fumarylacetoacetate hydrolase-deficient (Fah(-/-)) mouse, we studied the senescence of hepatocytes that had undergone continuous cell proliferation over a long time period, up to 12 rounds of serial transplantations. We demonstrated that the continuously proliferating hepatocytes avoided senescence and always maintained a youthful state. The reactivation of telomerase in hepatocytes after serial transplantation correlated with reversal of senescence. Moreover, senescent hepatocytes harvested from aged mice became rejuvenated upon serial transplantation, with full restoration of proliferative capacity. The same findings were also true for human hepatocytes. After serial transplantation, the high initial proportion of octoploid hepatocytes decreased to match the low level of youthful liver. CONCLUSION: These findings suggest that the hepatocyte "ploidy conveyer" is regulated differently during aging and regeneration. The findings of reversal of hepatocyte senescence could enable future studies on liver aging and cell therapy.

The double-edged effects of IL-6 in liver regeneration, aging, inflammation, and diseases.

Interleukin-6 (IL-6) is a pleiotropic cytokine and exerts its complex biological functions mainly through three different signal modes, called cis-, trans-, and cluster signaling. When IL-6 binds to its membrane or soluble receptors, the co-receptor gp130 is activated to initiate downstream signaling and induce the expression of target genes. In the liver, IL-6 can perform its anti-inflammatory activities to promote hepatocyte reprogramming and liver regeneration. On the contrary, IL-6 also exerts the pro-inflammatory functions to induce liver aging, fibrosis, steatosis, and carcinogenesis. However, understanding the roles and underlying mechanisms of IL-6 in liver physiological and pathological processes is still an ongoing process. So far, therapeutic agents against IL­6, IL­6 receptor (IL­6R), IL-6-sIL-6R complex, or IL-6 downstream signal transducers have been developed, and determined to be effective in the intervention of inflammatory diseases and cancers. In this review, we summarized and highlighted the understanding of the double-edged effects of IL-6 in liver homeostasis, aging, inflammation, and chronic diseases, for better shifting the "negative" functions of IL-6 to the "beneficial" actions, and further discussed the potential therapeutic effects of targeting IL-6 signaling in the clinics.

Interferon-α stimulates DExH-box helicase 58 to prevent hepatocyte ferroptosis.

BACKGROUND: Liver ischemia/reperfusion (I/R) injury is usually caused by hepatic inflow occlusion during liver surgery, and is frequently observed during war wounds and trauma. Hepatocyte ferroptosis plays a critical role in liver I/R injury, however, it remains unclear whether this process is controlled or regulated by members of the DEAD/DExH-box helicase (DDX/DHX) family. METHODS: The expression of DDX/DHX family members during liver I/R injury was screened using transcriptome analysis. Hepatocyte-specific Dhx58 knockout mice were constructed, and a partial liver I/R operation was performed. Single-cell RNA sequencing (scRNA-seq) in the liver post I/R suggested enhanced ferroptosis by Dhx58hep-/-. The mRNAs and proteins associated with DExH-box helicase 58 (DHX58) were screened using RNA immunoprecipitation-sequencing (RIP-seq) and IP-mass spectrometry (IP-MS). RESULTS: Excessive production of reactive oxygen species (ROS) decreased the expression of the IFN-stimulated gene Dhx58 in hepatocytes and promoted hepatic ferroptosis, while treatment using IFN-α increased DHX58 expression and prevented ferroptosis during liver I/R injury. Mechanistically, DHX58 with RNA-binding activity constitutively associates with the mRNA of glutathione peroxidase 4 (GPX4), a central ferroptosis suppressor, and recruits the m6A reader YT521-B homology domain containing 2 (YTHDC2) to promote the translation of Gpx4 mRNA in an m6A-dependent manner, thus enhancing GPX4 protein levels and preventing hepatic ferroptosis. CONCLUSIONS: This study provides mechanistic evidence that IFN-α stimulates DHX58 to promote the translation of m6A-modified Gpx4 mRNA, suggesting the potential clinical application of IFN-α in the prevention of hepatic ferroptosis during liver I/R injury.

[Electroacupuncture of acupoints of heart meridian ameliorates acute myocardial ischemia via Akt/mTOR pathway].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of myocardial protein kinase B (Akt) and mammalian target of rapamycin (mTOR) in acute myocardial ischemia (AMI) rats. METHODS: Thirty male SD rats were randomly divided into control, model and EA groups (n=10 in each group). The AMI model was established by occlusion of the descending anterior branch (DAB) of the left coronary artery. EA (2 Hz, 1-2 mA) was applied to bilateral "Shenmen" (HT7) and "Tongli" (HT5) for 20 min, once daily for consecutive 7 days. The electrocardiogram (ECG) of nape-xiphoid lead was recorded for assessing changes of myocardial ischemia. Histopathologic changes of the ischemic myocardial tissue were observed after H.E. staining and ultra-microstructural changes of cardiomyocytes observed by transmission electron microscopy (TEM). The expression levels of Akt, phosphorylated-Akt (p-Akt), mTOR and phosphorylated-mTOR (p-mTOR) in the myocardium were detected by Western blot, followed by calculating the ratios of p-Akt/Akt and p-mTOR/mTOR. RESULTS: Following ligature of DAB, the ECG-ST level was significantly increased in the model group in comparison with the control group (P<0.01). At 30 min after treatment, the ECG-ST level decreased significantly compared with the model group (P<0.01). At the end of the 7-day treatment period, the ECG-ST level increased compared with the model group (P<0.05). The levels of myocardial p-Akt and p-mTOR protein expression, and the ratios of p-Akt/Akt and p-mTOR/mTOR were significantly lower in the model group than those in the control group (P<0.01), and considerably increased in the EA group than in the model group (P<0.01). No significant differences were found among the three groups in the expression levels of Akt and mTOR proteins (P>0.05). Outcomes of H.E. staining and TEM showed damage of mitochondria and occurrence of a large number of autophagosomes in myocardiocytes in the model group, which was milder in the EA group. CONCLUSION: EA at HT5 and HT7 can improve AMI in AMI rats, which may be related to its effect in facilitating Akt/mTOR signaling.

[Both fumigation and heating of moxibustion promote wound healing and tissue repair in rats with full-thickness skin incision].

OBJECTIVE: To investigate the effect of moxibustion on wound healing, new capillaries and tissue repair in rats with full thickness skin excision. METHODS: SD rats were randomly divided into control, moxa-fumigating and moxa-heating groups, with 24 rats in each group. The full-thickness skin excision injury model was replicated according to Balaji's and colleague's methods. Rats in the moxa-fumigating group received fumigation of moxa-smog at the wound surface (25 min/time) immediately after modeling through a smoke-heat separation device, and those of the moxa-heating group received conventional thermal stimulation of the ignited moxa over the wound (25 min/time) without moxa smog, once a day for 10 consecutive days. The wound color, pus, carrion, granulation, and healing status of the rats were observed. H.E. staining and Masson staining were used to observe the local tissues and collagen fibers of the wound on the 3rd and 7th days. Newly born capillaries at the wound surface were observed on the 10th day. RESULTS: Following modeling, the wound area of the moxa-fumigating group on the 1st and 7th day, and those of both moxa-fumigating and moxa-heating groups on the 3rd and 5th day were significantly reduced compared with the model group (P<0.001), and the wound area of the moxa-fumigating group was obviously smaller than that of the moxa-hea-ting group (P<0.01，P<0.001). On the 3rd and 7th day after modeling, the re-epithelialization rates were significantly increased (P<0.001), and the amounts of inflammatory cells were significantly reduced in both moxa-fumigating and moxa-heating groups (P<0.001). At the same time, the re-epithelialization rate of the moxa-fumigating group was higher than that of the moxa-heating group (P<0.05，P<0.001), and the inflammatory cell count of the moxa-fumigating group was lower than that of the moxa-hea-ting group (P<0.001). On the 10th day after the modeling, the number of new capillaries and capillary density in the wound basal layer were significantly increased in both moxa-fumigating and moxa-heating groups (P<0.05, P<0.001, P<0.01), and were notably higher in the moxa-fumigating group than in the moxa-heating group (P<0.01, P<0.05). CONCLUSION: Both fumigation and heating of moxibustion can promote wound healing in rats with full-thickness skin excision injury，which may be related to their effects in controlling the inflammatory response and promoting the production of collagen fibers, granulation tissue and capillaries.

Aged-related Function Disorder of Liver is Reversed after Exposing to Young Milieu via Conversion of Hepatocyte Ploidy.

Previous study showed that senescent hepatocytes from aged liver could be rejuvenated after repopulated in the young recipient liver. The proliferative capacity of hepatocytes was restored with the senescence reversal. However, it is unknown whether metabolic and homeostatic function of aged liver, as well as age-dependent liver steatosis could be rejuvenated or alleviated. Here, we found that senescent hepatocytes from aged liver were rejuvenated after exposing to young blood. An autonomous proliferation of senescent hepatocytes which resulting in ploidy reversal might be the underlying mechanism of senescent reversal. After performing 2/3 partial hepatectomy (2/3PHx) in young blood exposed old liver, delayed DNA synthesis of senescent hepatocytes was rescued and the number of BrdU positive hepatocytes was restored from 4.39±2.30% to 17.85±3.21%, similarly to that in the young mice at 36 hours post 2/3PHx. Moreover, Cyclin A2 and Cyclin E1 overexpression of hepatocytes in aged liver facilitating the G1/S phase transition was contributed to enhance liver regeneration. Furthermore, lipid droplet spread widely in the elderly human liver and old mouse liver, but this aged-associated liver steatosis was alleviated as senescence reversal. Collectively, our study provides new thoughts for effectively preventing age-related liver diseases.

[Effect of different concentrations of moxa smoke exposure on nasal mucosal injury and expression of serum IL-1, IL-6 and TNF-α in rats].

OBJECTIVE: To observe the effect of long-term moxa smoke exposure of different concentrations on olfactory function in rats, and provide experimental basis of safety study of moxa smoke produced by moxibustion. METHODS: Forty SD rats were randomly divided into a normal control group, a low-concentration moxa smoke group, a moderate-concentration moxa smoke group and a high-concentration moxa smoke group, 10 rats in each one. The rats in the moxa smoke groups were put into three plexiglass moxibustion boxes with different moxa smoke concentrations, 4 hours per times, twice a day for 90 days. The general state of rats was evaluated before and during the experiment. After the intervention, the olfactory function was evaluated by two-bottle experiment (TBE); the morphology of nasal mucosa was observed by HE staining; the apoptosis of olfactory epithelial cells in nasal mucosa was detected by TUNEL method; the serum levels of interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected by ELISA method. RESULTS: In the late stage of moxa smoke exposure (45-90 days into intervention), the behavioral activity of rats in the moderate-concentration moxa smoke group and the high-concentration moxa smoke group was weaker than that in the normal control group, and their response to stimulation was strong, and their mental state was worse. After intervention, the drinking rate of vinegar-water mixture in the moderate-concentration moxa smoke group and the high-concentration moxa smoke group was higher than that in the normal control group and the low-concentration moxa smoke group (P<0.01). The hierarchical structure of nasal mucosa in the moderate-concentration moxa smoke group and the high-concentration moxa smoke group was unclear, disordered, necrotic and inflammatory cell infiltration was serious; the number of apoptotic cells in olfactory epithelium of nasal mucosa in the moderate-concentration moxa smoke group and the high-concentration moxa smoke group was more than that in the normal control group and the low-concentration moxa smoke group (P<0.01), that in the high-concentration moxa smoke group was more than the moderate-concentration group (P<0.01). The serum levels of IL-1, IL-6 and TNF-α in the low-concentration moxa smoke group, the moderate-concentration moxa smoke group and the high-concentration moxa smoke group were higher than the normal control group (P<0.01), and those in the moderate-concentration moxa smoke group and the high-concentration moxa smoke group were higher than the low-concentration moxa smoke group (P<0.01), and those in the high-concentration moxa smoke group were higher than moderate-concentration moxa smoke group (P<0.01). CONCLUSION: The long-term exposure to low, moderate and high concentrations of moxa smoke could cause pathological changes in nasal mucosa and increase the serum levels of IL-1, IL-6 and TNF-α; the moderate and high concentrations of moxa smoke exposure could cause a series of damage to olfactory function and reduce olfactory sensitivity in rats.

Inhibition of SIRT1 Limits Self-Renewal and Oncogenesis by Inducing Senescence of Liver Cancer Stem Cells.

PURPOSE: Cancer stem cells (CSCs) have been considered involving in tumorigenesis, local recurrence, and therapeutic drug resistance of hepatocellular carcinoma (HCC). To investigate novel and effective methods for targeting hepatic CSCs is crucial for a permanent cure of liver cancer. METHODS: The expression level of SIRT1 was detected in CSCs of HCC tissues and cancer cell lines. Expression of CSC markers, the self-renewal and tumorigenic ability of liver CSCs were analyzed with SIRT1 inhibition. Cellular senescence-related markers were used to detect CSCs senescence after inhibition of SIRT1. RESULTS: SIRT1 was highly expressed in CSCs of HCC cell lines and human HCC tissues. In vitro study revealed that decreasing of SIRT1 level significantly downregulated the stemness-associated genes of liver CSCs and reduced the CSC stemness properties. Also, downregulated SIRT1 suppressed liver CSCs proliferation by decreasing their self-renewal abilities. Furthermore, CSCs with decreased SIRT1 expression showed limited tumorigenicity and formed smaller HCC tumor in vivo. And SIRT1 decreased CSCs became more susceptible to chemotherapeutic drugs. Mechanistically, SIRT1 decreased CSCs became senescence through the activation of p53-p21 and p16 pathway. The data further indicated that the tumor formed from SIRT1-knockdown CSCs exhibited higher senescence-associated ß-galactosidase (SA-ß-Gal) activity but lower proliferative capacity. CONCLUSION: Taken together, these findings pointed that induction of senescence in liver CSCs is an effective tumor suppression method for HCC, and SIRT1 may be served as a promising target for HCC treatment.

[Design and application of a moxibustion device with separated moxibustion smoke and heat].

In the view of the defects of the commonly used moxibustion instruments and moxa heating instruments, such as the moxa ash cannot be removed automatically, the temperature of moxibustion and moxibustion smoke is difficult to be stabilized and adjusted, and the instruments are complex and expensive, a moxibustion device with separated moxibustion smoke and heat is designed. This device can automatically remove the moxa ash and keep it on the isolation net; the temperature of the moxibustion outlet is maintained at 43-48 ℃ (effective moxibustion temperature) for more than 40 minutes, and there is no visible moxa smoke; the temperature of the moxa smoke outlet is controlled between 28-75 ℃, and the effective discharge of moxa smoke can be realized without external power equipment. This device has the advantages of stable and controllable temperature of moxibustion outlet and moxa smoke outlet, automatic removal and collection of moxa ash, separation of moxa smoke without additional power, etc., which can be used in clinical and animal experiments for moxa heating, moxa smoke removal, etc.

[Influence of moxibustion with moxa sticks with different diameters and at different distances from surface of "Zusanli"(ST36) on local skin temperature in rats].

OBJECTIVE: To observe the impact of moxa-stick with different diameters and at different distances on skin temperature in local "Zusanli"(ST36) region, so as to select suitable specifications for moxibustion. METHODS: A total of 120 male SD rats were randomly divided into diameters of 0.5, 0.9, 1.2 and 1.8 cm, and distances of 1, 2, 3, 4 and 5 cm groups, with 6 rats in each group. Moxa-stick with different diameters mentioned above was applied to the right ST36 (right hind limb) for 10 min every time at different distances (between the ignited moxa-stick tip and the skin) mentioned above, and the left ST36 was used as the control point. The skin temperature was detected by using an infrared thermometer. RESULTS: After application of moxibustion to ST36 region, the skin temperature was increased gradually along with the increased diameter of moxa-sticks and decreased along with the increased distance from the ignited moxa-stick tip to the skin. There were no significant changes in the skin temperature of the left control acupoint ST36. The skin temperature was below 40 ℃, between 43 to 55 ℃, over 43℃ and between 43 to 61 ℃, when the moxa-stick was 0.5 cm, 0.9 cm, 1.2 cm and 1.8 cm in diameter, and was kept 1, 2, 3 and 3 to 5 cm away from the skin surface, respectively. When the moxa-stick with a diameter of 1.8 cm was kept at a distance of 1 to 2 cm, the skin temperature reached 71 to 93 ℃ to cause obvious local burn lesion. CONCLUSION: During moxibustion, the ignited moxa-sticks with diameters of 0.5, 0.9, 1.2 and 1.8 cm are suitable to be kept less than 1, 1 to 2, 2 to 3, and 3 to 5 cm away from the skin surface of ST36, respectively.

A Novel Rhynchophylline Analog, Y396, Inhibits Endothelial Dysfunction Induced by Oxidative Stress in Diabetes Through Epidermal Growth Factor Receptor.

Aims: Endothelial dysfunction appears in early diabetes mellitus partially because of epidermal growth factor receptor (EGFR) abnormal activation and downstream oxidative stress. The aim of this study was to determine whether Y396, a synthesized analog of rhynchophylline, could protect against endothelial dysfunction in diabetes and the underlying molecular mechanism. Results: Y396 could directly target the EGFR and inhibit its phosphorylation induced by high glucose and EGF, downstream translocation to the nucleus of E2F1, and its transcriptional activity and expression of Nox4. Diabetes-induced endothelium malfunction was ameliorated by Y396 treatment through EGFR inhibition. Downstream oxidative stress was decreased by Y396 in the aortas of type 1 diabetes mellitus mice and primary rat aorta endothelial cells (RAECs). Y396 could also ameliorate tunicamycin-induced oxidative stress in the aorta and RAECs. In addition, we again determined the protective effects of Y396 on high-fat diet/streptozotocin-induced type 2 diabetes mellitus. Innovation: This is the first study to demonstrate that Y396, a novel rhynchophylline analog, suppressed high-glucose-induced endothelial malfunction both in vivo and in vitro by inhibiting abnormal phosphorylation of EGFR. Our work uncovered EGFR as a novel therapeutic target and Y396 as a potential therapy against diabetes-induced complication. Conclusion: Y396 could directly bind with EGFR, and inhibit its phosphorylation and downstream E2F1 transcriptional activity. It could also preserve tunicamycin-evoked endothelial dysfunction and oxidative stress. It could protect against diabetes-induced endothelium malfunction in vivo through EGFR inhibition and downstream oxidative stress. Antioxid. Redox Signal. 32, 743-765.

Rejuvenating Strategies of Tissue-specific Stem Cells for Healthy Aging.

Although aging is a physiological process, it has raised interest in the science of aging and rejuvenation because of the increasing burden on the rapidly aging global population. With advanced age, there is a decline in homeostatic maintenance and regenerative responsiveness to the injury of various tissues, thereby contributing to the incidence of age-related diseases. The primary cause of the functional declines that occur along with aging is considered to be the exhaustion of stem cell functions in their corresponding tissues. Age-related changes in the systemic environment, the niche, and stem cells contribute to this loss. Thus, the reversal of stem cell aging at the cellular level might lead to the rejuvenation of the animal at an organismic level and the prevention of aging, which would be critical for developing new therapies for age-related dysfunction and diseases. Here, we will explore the effects of aging on stem cells in different tissues. The focus of this discussion is on pro-youth interventions that target intrinsic stem cell properties, environmental niche component, systemic factors, and senescent cellular clearance, which are promising for developing strategies related to the reversal of aged stem cell function and optimizing tissue repair processes.

Generation of insulin-secreting cells from mouse gallbladder stem cells by small molecules in vitro.

BACKGROUND: Stem cell-derived pancreatic ß-like cells hold great promise for treating diabetes. Gallbladder belongs to the extrahepatic bile duct system and possesses stem-like cells. These stem cells could be expanded in vitro and have the potential of differentiating into hepatocytes, cholangiocytes, or pancreatic cells. As the gallbladder is highly available, gallbladder stem cells provide a new cell source of pancreatic ß-like cells. In this study, we aimed to investigate an approach for the generation of pancreatic ß-like cells from gallbladder stem cells (GSCs) without genetic modification. METHODS: A CK19CreERT;Rosa26R-GFP mouse was used to isolate CK19+ cells, which represented EpCAM+ stem cells in the gallbladder. They were cultured in the modified Kubota's medium for expansion and further analyzed. Then, we developed a strategy to screen a combination of small molecules that can generate insulin-secreting cells from gallbladder stem cells. These cells were identified with markers of pancreatic cells. Finally, they were seeded into the cellulosic sponge and transplanted to the diabetic mice for functional examination in vivo. RESULTS: Gallbladder stem cells could be expanded for more than 15 passages. They expressed typical hepatic stem cell markers including CK19, EpCAM, Sox9, and albumin. By screening method, we found that adding Noggin, FR180204, and cyclopamine could efficiently induce gallbladder stem cells differentiating into insulin-secreting cells. These cells expressed Pdx1, Nkx6.1, and insulin but were negative for Gcg. After transplantation with the cellulosic sponge, they could ameliorate hyperglycemia in the diabetic mice. CONCLUSION: This study provides a new approach which can generate insulin-secreting cells from the gallbladder without genetic modification. This offers an option for ß cell therapy in treating type 1 diabetes.

Liver-enriched activator protein 1 as an isoform of CCAAT/enhancer-binding protein beta suppresses stem cell features of hepatocellular carcinoma.

PURPOSE: Liver cancer stem cells (CSCs) are known to be associated with the development, survival, proliferation, metastasis, and recurrence of liver tumors. The aim of this study was to investigate the association of liver-enriched activator protein 1 (LAP1) with hepatocellular carcinoma (HCC) and liver CSCs (LCSCs) and explore the impact of LAP1 on LCSCs. MATERIALS AND METHODS: Differences in LAP1 expression in liver cancer tissues versus matched para-tumoral liver tissues and LCSCs versus non-CSCs were analyzed by Western blotting, real-time polymerase chain reaction, immunohistochemistry, and flow cytometry. The effect of LAP1 on liver cancer cells was evaluated by the expression of CSC markers, oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle distribution and the number of apoptotic cells were analyzed to assess cell cycle and cell apoptosis. Furthermore, a mouse subcutaneous tumor implant model was established to explore the role of LAP1 in the development of HCC in vivo. Finally, the expression of CSC markers in paraffin-embedded sections was evaluated by immunofluorescence. RESULTS: LAP1 was weakly expressed in HCC tumors and cell lines and even weaker in LCSCs. LAP1 inhibited the expression of stem cell-associated genes and reduced the abilities of oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle assay revealed that LAP1 induced G1/G0 arrest. Furthermore, LAP1 decreased subcutaneous tumor-formation ability and the expression of CSC markers and Ki67 in vivo. CONCLUSION: LAP1 suppressed the stem cell features of HCC, indicating that it possessed an antitumor effect in liver cancer, both in vitro and in vivo; therefore, LAP1 may prove to be a potential target in liver CSC-targeted therapy.

Insulin-like growth factor 2 is a key mitogen driving liver repopulation in mice.

Hepatocyte transplantation holds great promise as an alternative to orthotopic organ transplantation in the treatment of liver diseases. However, obtaining clinically meaningful levels of liver repopulation has not been achieved because the mechanisms regulating hepatocyte proliferation in recipient livers have not yet been well characterized. In the mouse model of Hereditary Tyrosinemia Type I, the fumarylacetoacetate hydrolase-deficient (Fah-/-) mouse, we found gradually increasing expression level of insulin-like growth factor 2 (IGF2) in the hepatocytes of host livers. Similarly, high levels of IGF2 were found in the livers of patients with deficient FAH activity. Recombinant IGF2 directly promotes proliferation of primary hepatocytes in vitro. Inhibition on IGF2 expression through the interruption of PI3K/Akt and MAPK pathways significantly reduced the level of liver repopulation in Fah-/- mice. Interestingly, treatment with IGF2 before hepatocyte transplantation generally improved the amount of liver repopulation seen in various mice models of liver injury. Altogether, these findings underscore the underlying mechanisms of therapeutic liver repopulation in Fah-/- mice, and indicate that IGF2 is a potential hepatocyte mitogen for liver cell transplantation therapies.

The extent of liver injury determines hepatocyte fate toward senescence or cancer.

It is well known that induction of hepatocyte senescence could inhibit the development of hepatocellular carcinoma (HCC). Until now, it is still unclear how the degree of liver injury dictates hepatocyte senescence and carcinogenesis. In this study, we investigated whether the severity of injury determines cell fate decisions between hepatocyte senescence and carcinogenesis. After testing of different degrees of liver injury, we found that hepatocyte senescence is strongly induced in the setting of severe acute liver injury. Longer-term, moderate liver injury, on the contrary did not result into hepatocyte senescence, but led to a significant incidence of HCC instead. In addition, carcinogenesis was significantly reduced by the induction of severe acute injury after chronic moderate liver injury. Meanwhile, immune surveillance, especially the activations of macrophages, was activated after re-induction of senescence by severe acute liver injury. We conclude that severe acute liver injury leads to hepatocyte senescence along with activating immune surveillance and a low incidence of HCC, whereas chronic moderate injury allows hepatocytes to proliferate rather than to enter into senescence, and correlates with a high incidence of HCC. This study improves our understanding in hepatocyte cell fate decisions and suggests a potential clinical strategy to induce senescence to treat HCC.

SPINK1, PRSS1, CTRC, and CFTR Genotypes Influence Disease Onset and Clinical Outcomes in Chronic Pancreatitis.

OBJECTIVES: Rare pathogenic variants in the SPINK1, PRSS1, CTRC, and CFTR genes have been strongly associated with a risk of developing chronic pancreatitis (CP). However, their potential impact on the age of disease onset and clinical outcomes, as well as their potential interactions with environmental risk factors, remain unclear. These issues are addressed here in a large Chinese CP cohort. METHODS: We performed targeted next-generation sequencing of the four CP-associated genes in 1061 Han Chinese CP patients and 1196 controls. To evaluate gene-environment interactions, the patients were divided into three subgroups, idiopathic CP (ICP; n = 715), alcoholic CP (ACP; n = 206), and smoking-associated CP (SCP; n = 140). The potential impact of rare pathogenic variants on the age of onset of CP and clinical outcomes was evaluated using the Kaplan-Meier model. RESULTS: We identified rare pathogenic genotypes involving the SPINK1, PRSS1, CTRC, and/or CFTR genes in 535 (50.42%) CP patients but in only 71 (5.94%) controls (odds ratio = 16.12; P < 0.001). Mutation-positive patients had significantly earlier median ages at disease onset and at diagnosis of pancreatic stones, diabetes mellitus and steatorrhea than mutation-negative ICP patients. Pathogenic genotypes were present in 57.1, 39.8, and 32.1% of the ICP, ACP, and SCP patients, respectively, and influenced age at disease onset and clinical outcomes in all subgroups. CONCLUSIONS: We provide evidence that rare pathogenic variants in the SPINK1, PRSS1, CTRC, and CFTR genes significantly influence the age of onset and clinical outcomes of CP. Extensive gene-environment interactions were also identified.

[Study on the biological function of dermal fibroblasts in the wounds of diabetic and non-diabetic rats with deep burns].

OBJECTIVE: To explore the changes of the biological function of dermal fibroblasts (FBs) in the wounds of diabetic and non-diabetic burned rats and the pathogenesis of impaired wound healing in diabetes. METHODS: 80 Sprague-Dawley (SD) rats weighing 220 g were randomly divided into control and STZ-induced diabetic groups, and then deep partial thickness scald involving 10% TBSA was reproduced in the two groups. The diabetic groups were randomized into pre-scalding, post-scalding day (PSD 3), PSD 7, PSD 14 and PSD 21 groups, with 6 rats in each group. Controls were also randomized into 5 groups. Skin specimens from the wound were harvested at each time point. Cell cycles of FBs were analyzed with flow cytometry. The amount of hydroxyproline in the skin tissue was assessed on 0, 3, 7, 14, and PSD 21. The type I and III collagens were determined by ELISA. The expression of alpha-SMA in the dermal fibroblasts of each group was assessed by immunohistochemistry method. Transmission electron microscopy was used to observe the ultrastructure changes of FBs. RESULTS: Compared with that in the normal rats, the percentage of the cells in G(0)/G(1) phase in the DM group was evidently lower on PSD 0 (65.79 +/- 5.24 vs 82.43 +/- 9.68, P < 0.01). After the scalding, the percentage of the cells in G(0)/G(1) phase in DM group was significantly higher (70.00 +/- 4.27 vs 42.04 +/- 12.96, on PSD 3, P < 0.01), meanwhile the percentage of S phase was remarkably lower than those in C group on 3, 7, 14, 21PSD (P < 0.05, P < 0.01). The amount of hydroxyproline in the diabetic skin tissue was obviously lower than those of the responding control groups before (0.72 +/- 0.06 vs 1.42 +/- 0.28, P < 0.01) and after burn injury (P < 0.01). Furthermore, the rate of I/III collagen on 7, 14 and PSD 21 was much higher in DM group than that in C group (P < 0.01). The expression of alpha-SMA in DM groups on PSDS 3, 7, 14 and PSD 21 was evidently lower than those of the controls (levels 10.28 +/- 3.99, C group 28.42 +/- 2.73, on PSD 14, P < 0.01), although that inclined to be heightened after burn injury. Ultrastructure changes of FBs in the wounds of diabetic rats could be observed, such as the outstretched endoplasmic reticulum, un development of Golgi's body, lackness of microtubule and microfilament, a sharp increase of cytolysosomes, and so on. CONCLUSION: The FB proliferation in the diabetic skin is abnormal, the synthetical ability of collagen is weakened, the expression of alpha-SMA is insufficient, the microtubule and microfilament is lack, and the number of cytolysosomes increases. The pathogenesis of impaired-wound healing in diabetics might be related with the above mentioned factors.

Hepatocyte polyploidization and its association with pathophysiological processes.

A characteristic cellular feature of the mammalian liver is the progressive polyploidization of the hepatocytes, where individual cells acquire more than two sets of chromosomes. Polyploidization results from cytokinesis failure that takes place progressively during the course of postnatal development. The proportion of polyploidy also increases with the aging process or with cellular stress such as surgical resection, toxic stimulation, metabolic overload, or oxidative damage, to involve as much as 90% of the hepatocytes in mice and 40% in humans. Hepatocyte polyploidization is generally considered an indicator of terminal differentiation and cellular senescence, and related to the dysfunction of insulin and p53/p21 signaling pathways. Interestingly, the high prevalence of hepatocyte polyploidization in the aged mouse liver can be reversed when the senescent hepatocytes are serially transplanted into young mouse livers. Here we review the current knowledge on the mechanism of hepatocytes polyploidization during postnatal growth, aging, and liver diseases. The biologic significance of polyploidization in senescent reversal, within the context of new ways to think of liver aging and liver diseases is considered.