Extracellular vesicles derived from bone marrow mesenchymal stem cells ameliorate chronic liver damage via microRNA-136-5p.

Chronic liver damage (CLD) encompasses a spectrum of conditions and poses a significant global health challenge, affecting millions of individuals. Currently, there is a deficiency of clinically validated therapeutics with minimal side effects. Emerging evidence underscores the significant potential of extracellular vesicles derived from bone marrow mesenchymal stem cells (BMSC-EVs) as a promising therapeutic method for CLD. This study aimed to evaluate the influence of BMSC-EVs containing microRNA-136-5p (BMSC-EVs-miR-136-5p) on macrophage polarization during chronic liver injury and elucidate the mechanisms associated with the GNAS/PI3K/ERK/STAT3 axis. Surface markers of BMSCs were detected via Immunofluorescent Staining. Subsequently, EVs were harvested from the BMSC culture medium. In vivo fluorescence imaging was employed to locate the BMSC-EVs. Additionally, fluorescence microscopy was used to visualize the uptake of DIR-labeled BMSC-EVs by RAW264.7 cells. Various methods were employed to assess the impact of BMSC-EVs on the expression levels of inflammatory factors (IL-1ß, IL-6, IL-10, and TNF-α), M1/M2 macrophage markers (iNOS and Arg-1), and members of inflammation-related signaling pathways (GNAS, PI3K, ERK, and STAT3) in RAW264.7 cells co-cultured with BMSC-EVs. Loss-of-function approaches targeting miR-136-5p in RAW264.7 cells were subsequently utilized to validate the role of BMSC-EVs-miR-136-5p. The Luciferase Reporter Assay indicates that GNAS was identified to be a target of miR-136-5p, and miR-136-5p demonstrating increased within BMSC-EVs compared to Raw264.7-EVs. BMSC-EVs-miR-136-5p mitigated CCl4-induced liver inflammation and improved liver function by Suppressing the GNAS/STAT3 Signaling. Notably, miR-136-5p suppressed lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. BMSC-EVs-miR-136-5p alleviates CLD by activating M2 polarization through the GNAS-mediated PI3K/ERK/STAT3 axis. Accordingly, the members of this axis may serve as therapeutic targets.

Lipopolysaccharide/Toll-like receptor 4 signaling pathway involved Qingdu decoction treating severe liver injury merging with endotoxemia.

OBJECTIVE: To investigate the protective effects and underlying mechanism of Qingdu decoction (QDD) on experimental rats with severe liver injury induced by thioacetamide (TAA). METHODS: A total of 40 Wistar rats were randomly divided into normal group (n = 10) and experimental group (n = 30). Rats were administrated the same content of saline in normal group. The rats in the experimental group were pretreated with TAA at dose of 12 mg/kg lasting 8 weeks, and from 9th week to 12th week, with TAA at concentration of 36 mg/kg. During the 9th week to 12th week period, the rats were randomly divided into three subgroups (n = 10 each) simultaneously based on the treatment categories: model group, lactulose (LA, 3.5 mL/kg) group and QDD (5.95 g/kg) group, orally once per day respectively. At the 12th week, the content of serum alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin (TBIL), endotoxin (ET) and tumor necrosis factor a (TNF-a) was detected by automatic biochemical analyzer. The plasma prothrombin time (PT), prothrombin time-international normalized ratio (PTR) and prothrombin time activity (PTA) were measured by automatic coagulation analyzer. The level of lipopolysaccharide (LPS)-binding protein (LBP), cluster differentiation 14 (CD14) and Toll-like receptor 4 (TLR4) expressions was measured by both western blot (WB) and real-time polymerase chain reaction (real-time PCR). RESULTS: Compared with the model group, hepatic morphology in the QDD group was improved under light microscope and transmission electron microscope; at the same time, the contents of serum ALT, AST, TBIL, ET and TNF-α, and level of LBP, CD14 and TLR4 expressions in liver tissues were significantly decreased compared with the model group (P < 0.05), while PTA in the QDD group was enhanced (P < 0.05). CONCLUSION: QDD has the functional effect on improving the injured liver through inhibiting the LPS/TLR4 signaling pathway thus decreasing the level of the inflammatory medicators.

Protective effect of Yiguanjian decoction against DNA damage on concanavalin A-induced liver injury mice model.

OBJECTIVE: To investigate the inhibitory effect of Yiguanjian decoction (YD) on DNA damage in Concanavalin A (Con A)-induced liver injury mice model and to explain the possible mechanism. METHODS: METHODS: Totally 120 male BALB/c mice were randomly divided into 6 groups, 20 mice each: normal group, model group, Bifendate group, YD low dose group, YD middle dose group and YD high dose group. Except normal group, liver injury model induced by Con A was established. While modeling, each mouse in YD group was given YD (0.4 mL/20 g per day) by intragastric administration (0.13 g YD for YD low dose group; 0.26 g for YD middle dose group; 0.52 g for YD high dose group). Bifendate group was given Bifendate (0.2 g·kg-1·d-1) by gavage. Normal group and model group were fed with same volume of physiological saline daily. After 8 weeks, the serum alanine transaminase (ALT) and aspartate transaminase (AST) were tested. The hematoxylin-eosin staining was used to evaluate the grade of liver inflammation and liver fibrosis stage. Hepatocellular DNA damage was detected by single cell gel electrophoresis technology. The protein expression of tumor necrosis factor-α (TNF-α), Bax and MutT Homolog 1 (MTH1) was detected by western blotting and enzyme linked immunosorbent assay. Bax mRNA and MTH1 mRNA were detected by Real-time Polymerase Chain Reaction (PCR). RESULTS: YD can improve the degree of liver inflammation and fibrosis in the liver of chronic hepatitis mice, the dose effect relationship is remarkable (P < 0.05). YD can reduce liver cell DNA damage. The difference between YD middle dose group and model group was statistically significant (P < 0.05). YD middle dose group had decreased the protein expression of TNF-α in the mice liver of immunological liver injury (P < 0.05). YD can increase the protein expression of Bax (P < 0.05). Compared with normal group, the protein expression of MTH1 was decreased (P < 0.05), but there was no statistical significance between YD group and model group (P > 0.05). YD can increase the mRNA expression of Bax and MTH1 (both P < 0.05). CONCLUSION: YD can effectively inhibit the DNA damage in immunological liver injury mice, the mechanism may be that it can decrease the TNF-α and increase the Bax and MTH1 expression.

Changes of color and blood flow of the tongue in the mini-swine of immune hepatic injury.

OBJECTIVE: To investigate color and microvascular blood flow of the tongue in the mini-swine with immune hepatic injury. METHODS: Six Chinese mini-swine for experimental use, 3 males and 3 females, were randomly divided into two groups, normal group and model group, 3 swine in each group. The swine in the model group was administrated by injection of 5 mg/kg ConA into the vein of auricular back, once every other day, 3 times each week, for 2 weeks in total. The animal in the control group was administrated with equal volume of saline. At 9 o'clock in the morning of the 15th day of the experiment, each swine was anesthetized with intramuscular injection of 9 ml 2.5% pentobarbital sodium and 3 ml Maleate, and then picture of the tongue was taken, microvascular blood flow on the tongue and the liver was detected with a laser Doppler blood flowmeter; Blood was taken from the precaval vein. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (Tbil) and total protein (TP) were determined; Pathological changes of the liver and tongue tissues were investigated by means of HE staining; Serum TNF-alpha content was detected with ELISA assay. RESULTS: In the mini-swine with immune hepatic injury induced by ConA, the tongue color showed cyanotic color, microvascular perfusion in the liver and the tongue, and partial pressure of oxygen in the tongue tissue significantly decreased; and the microcirculatory perfusion of the tongue was significantly correlated with that of the liver and the HIS color spatial value of the tongue; Serum TNF-alpha content significantly increased. CONCLUSION: The mini-swine with immune hepatic injury induced by ConA conforms to pathological characteristics of immune hepatic injury. Formation of the cyanotic tongue is related with microcirculatory disturbance of the tongue, which can indirectly reflect hepatic microcirculatory state in the immune hepatic injury.