Application of sinomenine hydrochloride in promoting apoptosis of mesangial cells
A technology of sinomenine hydrochloride and mesangial cells is applied in the field of biomedicine to achieve the effect of promoting apoptosis and good clinical application prospect.
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Embodiment 1
[0027] Embodiment 1, screening autophagy inhibitor
[0028] 1. Main experimental materials
[0029] Serum and kidney tissue of mice. It can be separated and extracted on site before the experiment (the extraction method belongs to the routine experimental operation in this field), or it can be purchased from a commercial company.
[0030] 2. Experimental settings
[0031] Set different groups (control group, model group, drug group):
[0032] (1) normal control group;
[0033] (2) Adriamycin model group;
[0034] (3) IgA model group;
[0035] (4) Adriamycin model drug treatment group;
[0036] (5) IgA model drug treatment group;
[0037] 3. Experimental method
[0038] Take kidney tissue, cut into 0.5cm 3 Mix the small pieces with serum at a mass ratio of 1:5, put them in a petri dish, and set aside.
[0039] For the normal control group, normal saline was used to treat serum and kidney tissue. In the model group, serum and kidney tissue were treated with doxorubici...
Embodiment 2
[0041] Example 2, detection of autophagy inhibitors
[0042] 1. ELISA detection (5 groups × 1 indicator)
[0043] The level of TGF-β1 in the serum of each group was detected by ELISA method.
[0044] 2. Western Blot detection (5 groups × 3 indicators)
[0045] Kidney tissues from each group were collected, homogenized, and total protein was extracted. Western Blot was used to detect the related indicators of renal fibrosis, fibronectin (FN), laminin (laminin, LN), and autophagy-related protein LC3βII / I. Level.
[0046] 3. Pathological examination of renal tissue (5 groups × 3 indicators)
[0047] Kidney tissues were collected from each group, the renal cortex was separated, fixed with 10% formaldehyde, embedded in paraffin, stained with HE, PASM and Masson respectively, and made into thin sections for light microscope examination.
[0048] The result is as figure 1 As shown, in the kidney tissue of the normal control group, the glomerular structure was clear and complete,...
Embodiment 3
[0054] Example 3, experiment of sinomenine hydrochloride inhibiting cell autophagy
[0055] 1. Main experimental materials
[0056] Mouse kidney mesangial cells, sinomenine hydrochloride, TGF-β1 receptor inhibitor LY-2157299, autophagy inhibitor 3-MA, TGF-β1 ELISA detection kit, Smad2 / 3, p-Smad2 / 3, MMP- 2. Collagen type I, type III and type IV, as well as autophagy-related protein Beclin-1, primary antibody for Western Blot detection of LC3II / I, CCK-8 detection kit, etc.
[0057] 2. Experimental settings
[0058] Set different groups (control group, model group, drug group):
[0059] (1) normal control group;
[0060] (2) LPS model group;
[0061] (3) LPS model + TGF-β1 receptor inhibitor (LY-2157299, 1 μM) group;
[0062] (4) LPS model + sinomenine hydrochloride group;
[0063] (5) LPS model + autophagy inhibitor (3-MA, 2mM) group;
[0064] (6) LPS model + sinomenine hydrochloride + autophagy inhibitor (3-MA, 2mM) group.
[0065] 3. Experimental method
[0066] Inocu...
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