Green thorn fruit flavonoids on diabetic mice to pathological changes in liver tissue
【Abstract】 Objective To observe the blue thorn fruit flavonoids (FPR) in diabetic mice liver histopathological changes. Methods intraperitoneal injection of alloxan diabetic
mice given green thorn fruit flavonoids (300 mg * kg-1) ig
treatment, for 4 weeks. Respectively, at 2 weeks and 4 weeks in each group were randomly selected four were killed, the liver weighed, and then 4% paraformaldehyde. Liver paraffin
section, HE staining, microscopy. The results of diabetes mellitus (DM) index increased significantly in livers of mice liver cells lose the normal cord-like structure, narrow
sinusoids, liver cells, cord between the fiber cell proliferation. Liver cell swelling, granular degeneration occurs, the central veins of liver cells surrounding fatty degeneration, severe cases, liver cells were necrotic foci of multifocal, necrotic foci within the nuclear enrichment, fragmentation, dissolution, lymphocytes and neutrophils
infiltration. Central venous congestion and small veins. FPR treatment group, the liver index was significantly reduced liver cell cord-like structure clearly sinusoids than normal liver cells showed a slight granular degeneration. Conclusion
FPR diabetic mice can inhibit the enlargement of the liver, reducing liver pathological changes, effectively delay the diabetes caused by liver damage.
Key words diabetes, liver, blue gill morphology fruit flavonoids
Abstract: ObjectiveTo investigate the effect of
flavonoids from Prinsepia utilis Royle on the histomorphology of liver in diabetic mice.MethodsDiabetic mice induced by alloxan were given flavonoids from prinsepia utilis royle
orally 300 mg * kg-1 each day for four weeks. Two weeks and
four weeks later four mice in each group were randomly sacrificed, livers were taben out and the weights were measured, and then fixed with 4% polyoxym ethylene. The hepatic tissue was embedded with paraffin, sectioned, and stained with Hematoxylin and Eosin (HE), the histopathology was observed with microscope ~ ResultsCompared with normal control group, as for the model control group, hepatic index of mice markedly increased, liver cell cords were disordered or disappeared, liver antrum became more narrow, fibrocytes
proliferated between liver cell cords; hepatic cells were swollen and developed granular degeneration, hepatic cells around central vein developed fatty degeneration, some serious, hepatic cells appeared many necrotic areas, cellular
nucleus concentrated, fragmentated, dissolved in necrotic areas, lymphocytes and neutrophils infiltrated in necrotic areas; central vein and small vein stagnanted blood.While compared with model control group, in the treatment group, hepatic index of mice markedly decreased, liver cell cords clear, liver antrum almost recovered to normal, hepatic cells developed slightly granular degeneration.ConclusionFlavonoids from Prinsepia utilis Royle can inhibit hepatic hypertrophy , relieve the changes of hepatic pathology, and delay the
progression of injury of liver induced by diabetes mellitus.
Key words: Flavonoids from Prinsepia utilis Royle; Diabetes mellitus; Liver; Histomorphology
Diabetes (diabetes mellitus, DM) is a metabolic disorder as the main body of sugar, a variety of physical and metabolic disorders associated with multiple organ injury in systemic disease, its long-term high-sugar, hyperlipidemia is caused by the body organs important reason for injury. Chronic complications of diabetes include not only the kidneys, heart, retina, nerve lesions, the liver is also a long-term high
blood sugar, one involving the target organs, diabetes, persistent hyperglycemia can lead to liver disease , with the extension of duration of diabetes, place the risk of liver
disease and severity will increase accordingly, therefore, diabetes, liver disease state has drawn increasing attention [2 ~ 4]. In recent years, a large number of studies have shown that many flavonoids in the treatment of diabetes and its
complications effect is significant, such as puerarin, epimedium, mulberry leaves, celery, Huang Qin, guava and other
plants flavonoids extract .
Green thorn fruit Prinsepia utilis Royle., The Chinese species of the total spent bian core wood, Li subfamily of
nuclear bian wood species, deciduous shrub , in the "southern Yunnan Materia Medica" and known as the Green barbed tip, Yunnan Qujing area known as the Bayonet fruit, Lijiang Naxi known as the Arras; produced in Yunnan, Guizhou, Chongqing, Sichuan and Tibet. Green thorn fruit oil yield an average of 28%, fruit oil is rich in a variety of nutrients the body needs, is a high natural oil, and the treatment of high blood fat, lower cholesterol, prevent cardiovascular disease are important supplementary effect . Green thorn fruit flavonoids (flavonoids from Prinsepia utilis Royle, FPR) is extracted from the blue thorn fruit out of the active ingredient, through animal experiments and found a clear hypoglycemic effect on diabetic mice in order to explore the
pathological changes in liver tissue conducted the following studies.
1.1 Animals ICR mice, ?, weight (23 ? 2) g, were
purchased in Laboratory Animal Research Institute, Sichuan Academy of Medical Sciences. Mice were divided into caged at
room temperature (22 ? 1) ?, relative humidity (60 ? 5)%,
fed regular rat feed, free drinking water.
1.2 Subjects drugs and reagents green thorn fruit flavonoids (flavonoids from Prinsepia utilis Royle, FPR), by the laboratory to extract from the blue thorn fruit be, temporary use of the former with 0.2% sodium carboxymethyl cellulose paired with blue thorn fruit Flavonoids 15mg/ml of the suspension; alloxan (alloxan, ALX, Sigma Company), clinical use before the alloxan paired with 7.5% saline
1.3 Instrument Lelca slicer (Germany); Nikon Biological Microscope (Japan); Nikon camera (Japan); digital thermostat water bath pot (Guohua Electric Appliance Co., Ltd.); electronic balance (Shenyang Dragon Electronics Co., Ltd.).
2.1 The establishment of diabetic model mice 3 d after the first adaptive feeding, fasting 12 h, free water, each mouse by 200 mg * kg-1 intraperitoneal injection of ALX
produced a one-time DM mice. The first 4 days after injection,
measured fasting glucose, fasting plasma glucose value is greater than 10.00 mmol * L-1 were included in diabetic model
[8 and 9].
2.2 Animal grouping and drug delivery to diabetic mice were randomly divided into: the model group (model control
group, DM group) (no treatment group); Green thorn fruit flavonoid group (Flavonoids from Prinsepia utilis Royle group, FPR group); At the same time a separate normal group (normal control group, NC group); each day, fed regular rat feed, free
drinking water. Green thorn fruit flavonoid group to dose of 300 mg * kg-1, ig administration of 1 / d, model control group and normal group fed with normal saline 1 times / d, for 4 weeks.
2.3 Sample collection and processing of experiments 2 weeks and 4 weeks, mice in each group randomly selected four, weighing, the mice were sacrificed broken neck bleeding, rapid caesarean section to remove the liver, the filter paper suction net residual blood, liver, said that electronic balance weight, and then part of his liver placed in 4%
paraformaldehyde fixed, conventional methods of production, HE staining, light microscopy observation of pathological changes in the liver.
2.4 Statistical analysis of data by ? s said that all
the data using SPSS13.0 statistical package deal between the two groups tested with the Duncan method.
3.1 Green thorn fruit flavonoids in diabetic mice liver organ index in DM mice, the liver weight and liver index were significantly higher than NC mice, FPR focuses on four
weekends in liver was significantly lower than DM group, FPR group of liver index was significantly lower than that of the DM group of mice at the same time. Description FPR can reduce the liver index of diabetes. The results in Table 1. Table 1
diabetic mice liver index changes (slightly)
3.2 Green thorn fruit flavonoids in diabetic mice pathological changes of liver tissue
3.2.1 anatomy changes in NC group and normal liver, appearance smooth, wet, sharp edges, sub-leaf clear, normal
size, texture soft and elastic; DM visceral adhesion is very serious, liver enlargement, dull red color, texture, crisp liver margin blunt, lobular integration, sub-leaf is not
obvious. FPR groups of mice with the lesions to reduce the extension of treatment time.
NC groups: normal liver tissue (see Figure 1).
DM Group: 2 weekends, loss of normal liver cells, cord-
like structure, the liver cell swelling, granular degeneration occurs. Sinusoid narrowing, liver cells, a small amount of
fiber cable can be seen between the cell proliferation. Mild central venous congestion, a small amount of perivascular inflammatory cell infiltration (see Figure 2). Around the central vein of liver cytoplasm filled with lipid droplets of
varying sizes of round, showing fatty degeneration (Figure 3). 4 weekend, the liver cell cord-like structure completely
disappeared, the liver cell swelling aggravated sinusoid narrowing, liver cells, increased proliferation of fibroblasts between the cable, the central veins and small venous stasis more severe (see Figure 4), severe cases, liver cells were multifocal necrosis, necrotic foci within the nuclear enrichment, fragmentation, dissolution, lymphocytes and neutrophil infiltration (see Figure 5).
FPR Group: 2 over the weekend, the liver cells, cord-like
structure more clearly, the liver cells, swollen round, narrow sinusoids, liver cells can be seen a small amount of lymphocytic infiltration between cable and fiber cell proliferation (see Figure 6); 4 weekend, the liver cells, cord-like structure clearly sinusoids than normal liver cells showed a slightly granular degeneration (see Figure 7).
Glucose metabolism in the liver is one of the most important organs, mainly through the promotion of glycogen
synthesis and glucose utilization, inhibiting gluconeogenesis and other ways to lower blood glucose, while the liver glucose metabolism mainly in the insulin-induced complete. ALX-induced
DM mice decreased insulin levels, therefore, diabetes, glucose metabolism of mouse liver disorder, glycogen synthesis decreased glucose utilization decreased gluconeogenesis in, leading to hyperglycemia. In this experiment, we used a one-
time intraperitoneal injection of alloxan 200 mg * kg-1 made
DM model, 72 h after injection most of fasting blood glucose in mice were higher than 10.00 mmol * L-1, consistent with
reported in the literature [8, 9], and mice into a mold at a high level blood sugar steady state, no spontaneous ease the
trend shows that diabetes model is successful. Reposted elsewhere in the paper for free download http://
The experiment found that ALX diabetic mice caused significant enlargement of the liver, showing for the DM group
Mouse liver index were significantly higher than NC. Diabetic mice after treatment by the FPR, the liver index were significantly lower than the DM group. Survey shows a high incidence of diabetic hepatomegaly has been reported from 12% to 90%, if not a long-term control will be to develop
cirrhosis of the liver. Hepatomegaly, liver damage is one of the main lesion . Liver index is a measure of objective indicators of enlargement of the liver, it can rule out the weight of large animals with large individual differences in
the liver, as well as each animal's own liver increased with the weeks of growth factors, so that the growth of liver weight more objective parameters. FPR can reduce the liver index of diabetic mice, indicating FPR can inhibit the enlargement of the liver. Reported in the literature , blood sugar well-controlled diabetic patients and only 9% had hepatomegaly, without control, compared to 60%. Previous studies suggest that DM hepatomegaly is mainly due to liver cells caused by accumulation of fat or glycogen [12,13]. In
this study, DM was observed in mice liver cells filled with lipid droplets of varying sizes, indicating that this mouse model hepatomegaly due to liver cells caused by fat accumulation. DM caused by fatty liver due to: high blood
sugar caused by abnormal lipid metabolism, surrounded by decomposition of adipose tissue hyperthyroidism, increased blood lipids into the liver and free fatty acids increased, exceeding the scavenging ability of the liver, intrahepatic fat accumulation of fatty liver occurs. FPR inhibit
enlargement of the liver may be due to its hypoglycemic effect related to the early stage we have already conducted a hypoglycemic FPR trials in the "blue thorn fruit Flavonoids on Alloxan-induced diabetic mice hypoglycemic effect" in this
article reported that FPR has good hypoglycemic effect, FPR by lowering blood glucose to free fatty acid reduction in liver cells almost no accumulation of lipid droplets, hepatomegaly significantly eased.
DM mice liver 4 weekend noticeable multifocal necrosis
accompanied by a large number of inflammatory cell infiltration, the reason may be related to an increase in the DM state, free radicals. High glucose condition within the organization's own glucose oxidation, and protein non-
enzymatic glycation to form advanced glycation end products (advanced glycation end products, AGEs) in the process produce large amounts of free radicals. The liver is rich in superoxide dismutase, can effectively remove free radicals produced by the body to prevent or inhibit lipid peroxidation, however, the non-enzymatic protein glycosylation so that
inactivation of free radical scavenging enzymes, free radical scavenging capacity weakened. An increase in the amount of free radicals and free radical scavenging ability of weakened,
leading to liver cell membrane and mitochondria of liver cells will be subject to free radical damage , biofilm is a unsaturated fatty acid composition of lipids and proteins, membranes and organelles are for the biofilm structure. When a
large number of free radicals acting on the membrane phospholipids of unsaturated fatty acids in lipid peroxidation occurred, so that can regulate membrane fluidity of unsaturated fatty acids reduce membrane fluidity decrease, while the saturated fatty acids makes membranes become rigid. The injury also caused mitochondrial respiratory chain enzyme inactivation, H-ATPase structural integrity being compromised, decreased synthesis of liver cells, insufficient energy supply due to necrosis. Lipid peroxidation can also be caused by
membrane permeability increase, induced by a large number of extracellular Ca2 flow, resulting in swelling of mitochondria, lysosomes release. Free radicals can also cause denaturation
of protein inactivation, nucleic acid and chromosome damage
induced cell death. In addition, free radicals also by activating phospholipase catalytic membrane phospholipids with the release of leukocyte chemotactic effect of arachidonic acid and platelet-activating factor, so that a large number of
white blood cell aggregation increase in local inflammation. In addition, lipid droplets accumulated in the liver cells can also result in further necrosis of liver cells . FPR necrosis can heal, the reason may be related to the hypoglycemic effect, by lowering blood glucose and free fatty acids reduce the production of free radicals, thereby reducing the freedom of self-and free fatty acids on liver cell damage, the precise mechanism has yet to be studied.
The DM model for other major lesions in livers of mice
fibroblast proliferation, after treatment by the FPR, fiber cells decreased. Currently considered the promotion of high blood glucose has an important role in liver fibrosis [16 ~ 18]. High blood sugar caused by the formation of advanced
glycation end products, leading to a series of cytokines, such as connective tissue growth factor (connective tissue growth
factor CTGF), transforming growth factor-β (TGF-β), by these
cytokines to promote liver fibrosis. Hyperglycemia may cause increased expression of fibroblast CTGF, thereby contributing to hepatic fibrosis . TGF-β can induce specific product
of the CTGF-induced fibrosis . In addition, high blood sugar can cause oxidative stress, oxidative stress by generating a series of inflammatory mediators, induce CTGF produced the promotion of liver fibrosis . FPR to reduce fibroblast proliferation might be related to lowering blood glucose related to lower blood glucose by reducing the production of cytokines, thereby reducing the proliferation of fibroblasts, a specific part of what the mechanism remains to be further studied.
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