| Part one I dentification of components and determination of some effective components in Mingmu Xiaomeng tabletsObjectiveTo identify the components of Mingmu Xiaomeng tablets and detect the content of some effective compounds?MethodsBased on the the MS/MS spectrometry information of compounds,and combined with the mass spectrometry data of standard compound,relative databases and literatures,the constituents of Mingmu Xiaomeng tablets were identified and the contents of 11 effective compounds were detected by using the technology of ultra-high performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry(UPLC-Q-Orbitrap HRMS).ResulisA total of 49 compounds were identified,including flavonoids,organic acids,alkaloids,saponins and so on.The presence of these compounds further confirmed by the content detection of 11 compounds.ConclusionUPLC-Q-Orbitrap HRMS technology can identify various chemical components in Mingmu Xiaomeng tablet systematically,accurately and efficiently,and also provide an analytical method for the content detection of active components,which provides a scientific theoretical basis for studying its active component identification,quality control,potential mechanism of action and guiding clinical rational use of drugs.Part two Effect of Mingmu Xiaomeng tablet on the turbulence of retinal autophagy in diabetic ratsObjectiveTo investigate the effect of Mingmu Xiaomeng tablet on the expression of autophagy related proteins in retina of diabetic rats.MethodsThirty-five male SD rats were randomly divided into normal control group,diabetic model control group,and low,medium and high dose gavage group of Mingmu Xiaomeng tablet.The rat model of diabetes was established by intraperitoneal injection of STZ 60mg/kg dose in the other four groups except the normal control group.After one week of successful establishment of the diabetic model,the rats'general condition and changes of body weight and fasting blood glucose were observed.The expression of au tophagy protein LC3-? and p62 in rat retina was detected by western blot after 14 weeks and HE staining was used to observe the retinal tissue morphology in each group.Resulis1.Three days after injection of STZ to the end of the experiment,the weight of diabetic model control group was lower than that of the normal control group(P<0.05);compared with the diabetic model control group,the weight of the gavage group of Mingmu Xiaomeng tablet was higher than that of the diabetic model control group 2 weeks after modeled,and the low dose and high dose gavage group of 8 weeks,10 weeks,12 weeks,14 weeks had statistical difference(P<0.05).2.The blood glucose of rats in the normal control group was always maintained at the normal level between 4 mmol/L-7mmol/L;the blood glucose in the model group was higher than that in the normal control group(P<0.05),and there was no statistical difference between the blood glucose in the gavage group of Mingmu Xiaomeng tablet and the diabetic model control group(P>0.05).3.Compared with the normal control group,the expression level of LC3-? and p62 in the retina of the diabetic model control group increased,the difference was statistically significant(P<0.05).Compared with the diabetic model control group,the expression level of LC3-? and p62 in the treated group of Mingmu Xiaomeng tablet decreased,among which the medium dose and high dose group had statistical significance(P<0.05).4.In the normal control group,the layers of retina were arranged orderly and clearly.In the diabetic model control group,the retinal nerve fiber layer was thickened and edematous;the number of ganglion cells was decreased,the structure of ganglion cell layer was loose and disordered,with vacuole like changes;the inner plexiform layer had obvious edema,with glial cell proliferation;the arrangement of the inner layer was disordered and loose,with vacuole like degeneration;the arrangement of the outer layer was disordered and loose,with large intercellular space;The photoreceptor cells are arranged in filaments,and are still in order,with vacuole like changes.Dilation of capillaries and hyperemia can be observed in some cases.In the Mingmu Xiaomeng tablet group,the structures of retina layers were orderly arranged with clear layers;the nerve fiber layer was partially thickened with edema;the ganglion cell layer was partially vacuolated with decreased number;the inner plexiform layer was relatively complete with edema;the inner plexiform layer was orderly arranged with large nucleus,complete structure and deep staining;the outer plexiform layer was clearly visible;The cell bodies of the outer nuclear layer were arranged in order and dense,with deep staining;the inner and outer nodes of the photoreceptor cells were arranged in filaments,with a small amount of vacuole like degeneration.ConclusionThe level of LC3-? in diabetic model control group increased,which indicated that diabetes activated autophagy of retinal cells,but the autophagic flux was compromised,the downstream of autophagy was inhibited,lysosome was damaged,the degradation of autophagolysosome was reduced,and p62 was increased.After intragastric administration of Mingmu Xiaomeng tablets,the expression levels of p62 and LC3-? were decreased,which suggested that Mingmu Xiaomeng tablets could promote the reduction in autophagosome degradation by lysosome systems and promote the autophagy of retinal cells.Part three Effect of Mingmu Xiaomeng tablet on PI3K/Akt/mTOR pathwayObjectiveTo investigate the effect of Mingmu Xiaomeng tablet on the expression of PI3K/Akt/mTOR related protein and the level of inflammatory factor protein in serum of diabetic rats.MethodsThirty-two male SD rats were randomly divided into normal control group,diabetic model control group,Mingmu Xiaomeng tablet group and calcium dobesionate group.In addition to the normal control group,other rats made diabetes model according to the second part of the method.After 14 weeks,western blot was used to observe the expression of autophagy protein LC3-?,p62 and upstream pathway PI3K/Akt/mTOR were detected in the retina of rats in each group,and the effect of Mingmu Xiaomeng tablet on Muller cells in retina was determined by immunofluorescence and immunohistochemistry;the effect of Mingmu Xiaomeng tablet on retina function of rats was detected by ERG;the ultrastructure of retina in each group was observed by transmission electron microscopy;the effect of Mingmu Xiaomeng tablet on inflammatory factors in serum of rats was detected by protein chip technology.Results1.Compared with the normal control group,the expression level of LC3-? and p62 in the retina of the diabetic model control group increased,the difference was statistically significant(P<0.05);compared with the diabetic model control group,the level of LC3-? and p62 in the Mingmu Xiaomeng tablet and calcium dobesilate gavage group decreased,the difference was statistically significant(p<0.05).Compared with the normal control group,the expression of p-PI3K,p-Akt and p-mTOR was significantly up-regulated(p<0.05);Compared with the diabetic model control group,the expression of p-PI3K,p-Akt and p-mTOR protein in Mingmu Xiaomeng tablet group was significantly down regulated(p<0.05);the expression of p-Akt and p-mTOR protein in calcium dobesilate group was significantly down regulated(P<0.05).2.After three month of administration,compared with the normal control group,the red fluorescence intensity of GFAP in the nerve fiber layer,ganglion cell layer and the inner reticulum layer in the diabetic group was significantly enhanced.In the immunohistochemistry,brown long protuberances were seen throughout the inner and outer nuclear layer,showing the trend of GFAP expression throughout the entire layer of the optic reticulum.Compared with the diabetic model control group,GFAP positive red fluorescence staining was found in the nerve fiber layer and ganglion cell layer of the Mingmu Xiaomeng tablet group and the calcium dobesilate group,The fluorescence was weak,and the brown filamentous process also reduced in the immunohistochemistry.3.Compared with the normal control group,the amplitude of a wave,b wave and OPs2 wave in ERG of diabetic rats decreased gradually,and the difference was statistically significant(P<0.05).Compared with the diabetic model control group,the amplitude of a wave,b wave and OPs2 wave in Mingmu Xiaomeng tablet increased after gavage,and the amplitude of a wave and b wave were statistically different(P<0.05);the amplitude of a wave,b wave and OPs2 wave in calcium dobesilate gavage group increased,and the amplitude of b wave was statistically different(P<0.05).4.In the diabetic model control group,the swelling and vacuolation of mitochondria were found in the cytoplasm around the nucleus of Muller cells.In addition,the cytoplasm around the nucleus of other nerves in the nuclear layer was swollen,mitochondria were swollen,cristae were broken and vacuolated.The outer nuclear layer is arranged disorder,the nuclear membrane is sunken,and the intercellular space can be seen;the outer membrane disc of photoreceptor cells is arranged in disorder,loose and deformed,and breaks and dissolves,and the mitochondria are swollen,and giant mitochondria can appear,with obvious vacuolation.The basement membrane of capillaries is thickened and the lumen is narrow.In the gavage group of Mingmu Xiaomeng tablet,there was a small amount of mitochondrial edema and vacuolation in the cytoplasm around the nucleus of Muller cells in the nuclear layer,the outer nuclear layer was more orderly;the outer segment membrane disc of photoreceptor cells was orderly arranged,and the local area was loose,and there was no obvious mitochondrial swelling and vacuole like changes.The basement membrane of capillaries was uneven and the lumen was narrow.5.The results showed that compared with the normal control group,the expression of IL-1 ?,IL-4,IL-6,TNF-? and VEGF in the diabetic model control group was significantly increased,the difference was statistically significant(P<0.05).Compared with the diabetic model control group,the expression of IL-1?,IL-4,IL-6,TNF-?,VEGF in serum of Mingmu Xiaomeng tablet group decreased significantly(P<0.05),while the expression of IL-1?,IL-4,IL-6,TNF-?,VEGF in serum of calcium dobesilate group decreased significantly(P<0.05).Conclusion1.The results showed that the LC3-??p62 level and the expression of p-PI3K?p-Akt?p-mTOR protein in retinal tissue could be down-regulated in the gavage group of Mingmu Xiaomeng tablet,indicating that Mingmu Xiaomeng tablet could effectively inhibit the activation of PI3K/Akt/mTOR signaling pathway,promote the fusion of lysosome and autophagosome,and then enhance the autophagy level obviously.2.In the early stage of diabetes,retinal glial cells change,showing the reactive activation of glial cells dominated by Muller cells,which indicates that in the early stage of diabetes,retinal nerve cells have been injured due to the disorder of glucose metabolism.Mingmu Xiaomeng tablet can inhibit the high expression of retinal Muller cells in diabetic rats GFAP,and play a protective role in early diabetic retinal neurodegeneration.3.The amplitude of a wave and b wave of ERG maximum mixed reaction in diabetic model control group decreased gradually,and the amplitude of b wave decreased more obviously than that of a wave,indicating that in the early stage of DR,there were changes in the functions of photoreceptor cells,bipolar cells and Muller cells;the amplitude of OPs basically disappeared,indicating that the retina of diabetic model control group was seriously ischemic and hypoxic.The amplitude of a wave,b wave and OPs wave in diabetic rats increased after Mingmu Xiaomeng tablet was administrated to the stomach,which indicated that Mingmu Xiaomeng tablet had a protective effect on the retinal function of diabetic rats,which might be related to improving the blood circulation of retina.4.The serum protein inflammatory factors increased in the diabetic model control group,but decreased protein inflammatory factors after gavage of Mingmu Xiaomeng tablet.the result of this decrease may be related to the inhibition of PI3K/Akt/mTOR signaling pathway,thus reducing the release of inflammatory factors. |
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