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Polyprezinc | Treatment of small intestinal mucosal damage caused by non-steroidal anti-inflammatory drugs

Polyprezinc | Treatment of small intestinal mucosal damage caused by non-steroidal anti-inflammatory drugs

  • Categories:Stomach healthy
  • Author:
  • Origin:
  • Time of issue:2020-12-03
  • Views:0

(Summary description)

Polyprezinc | Treatment of small intestinal mucosal damage caused by non-steroidal anti-inflammatory drugs

(Summary description)

  • Categories:Stomach healthy
  • Author:
  • Origin:
  • Time of issue:2020-12-03
  • Views:0
Information

  Preface

  With the aging of society, the number of elderly and patients who need non-steroidal anti-inflammatory drugs has increased sharply, such as acetylsalicylic acid (ASA) and aspirin, which are widely used in various treatments due to their analgesic and anti-inflammatory effects. Inflammatory diseases. Although aspirin can cause gastric ulcers, based on studies of intestinal permeability and fecal inflammation markers, it is generally believed that aspirin will not cause any damage to the small intestine. However, with the application of capsule endoscopy, it is not impossible to find that aspirin induces small intestinal mucosal damage. Non-steroidal anti-inflammatory drugs cause repeated and severe gastrointestinal mucosal damage through a variety of mechanisms, including the reduction of prostaglandins, the activation of neutrophils, microcirculation disorders and the production of oxygen free radicals. Heat shock protein (HSP) is a type of protective protein with a variety of biological activities, and is involved in the protection of a variety of cell apoptosis, cancer and cytotoxic damage. Promoting the expression of HSP can protect cells from damage under various stresses or stimuli. HSP70 is a molecular chaperone, which is rapidly induced under physical and chemical stresses such as heat, oxidative stress, and drug exposure. The anti-apoptotic mechanism of HSP70 includes inhibition of c-Jun N-terminal kinase (JNK) signaling pathway, The activation of caspase, the release of mitochondrial cytochrome c and the formation of apoptosis suggest that high expression of HSP70 can reduce oxidative stress and cell damage, and has a strong cytoprotective effect. Therefore, drugs or treatments that induce the expression of HSP may play a positive role in the gastric mucosal defense system, and at the same time, participate in the cell protection of the gastric mucosa. Polyprezinc (PZ) is a chelate composed of zinc ions and L-carnosine, and is widely used clinically to treat gastric ulcers. The protective effect of PZ on mucosa is mainly attributed to its stimulation of mucus secretion, antioxidant activity, membrane stabilization and induction of HSP70. Studies have shown that PZ can protect the gastric mucosa from various stimuli both inside and outside the body. This article explored the relationship between PZ and ASA-induced apoptosis of rat intestinal epithelial cells RIE1, and clarified the role of PZ-induced HSP70 expression in this cell apoptosis.

  Materials and Method

  1.1 Cell lines and media

  Rat small intestinal epithelial cells (RIE1) were cultured in a humidified incubator at 37°C and 5% CO2, and the experiment was performed when the cells were fused.

  1.2 Cell viability

  Use cell lysis and screening fluorescence assay (FACLS) to measure cell viability, and measure the fluorescence intensity at Ex/Em=420/460nm.

  1.3 Microscopic analysis of apoptotic cells

  HO342 is a cell-permeable fluorescent dye with affinity for DNA, which can be used for nuclear morphology analysis to assess cell apoptosis. HO342 enters the cell with a complete cell membrane and stains DNA blue, while the polar compound PI only enters apoptotic or apoptotic cells with damaged cell membranes and stains DNA red. Therefore, living cells with cell membrane function, living cells and early apoptotic cells use blue dye (HO342), and red stained cells (PI) are late apoptosis or necrosis. Fluorescent cell nuclei were observed under an Olympus microscope with 40 times magnification using an inverted fluorescence microscope.

  1.4 Antibody and Western Blot

  "The main antibody specific for HSP70 (SPA-812) comes from stressor, and the anti-actin antibody (sc-10731) comes from a biological company. Immune complexes were detected by western blotting and quantified by Image J software.

  1.5 Small interfering RNA transfection

  The "negative control group (Mock-RIE1)" replaced HSP70-siRNA with 10 nM medium GC content siRNA (Invitrogen).

  1.6 Assess cell viability and apoptosis

  The annexin V/PI double staining method of annexin V-FITC apoptosis detection kit I was used to quantify cell apoptosis. FACS Calibur was analyzed with Cell Quest software.

  1.7 Statistical analysis

  All data are expressed as the mean (SEM) ± standard error, and the data adopts t test or one-way analysis of variance, and p<0.05 has significant difference.

  Results and analysis

  2.1 ASA induces RIE1 cell apoptosis

  "In order to determine the toxicity of ASA to RIE1 cells, the fused RIE1 cells were incubated with 0-15mM ASA for 24h. The results showed that ASA induced apoptosis in RIE1 cells in a dose-dependent manner. In addition, ASA (15mM) induced apoptosis in RIE1 cells in a time-dependent manner. Based on this result, we chose 15mM as the concentration of ASA, 15h as the culture time of RIE1 cells, and performed the following experiments.

  2.2 PZ inhibits RIE1 cell apoptosis induced by ASA

  PZ (70μM) pretreatment for 24h significantly inhibited ASA (15mM)-induced apoptosis of RIE1 cells, while PZ itself did not affect cell viability.

  2.3 PZ inhibits ASA-induced apoptotic dominant cell apoptosis

  To assess the type of apoptosis of RIE1 cells, the HO342 and PI double staining method was used. A small number of apoptotic cells were seen in the control group, including early apoptotic cells (blue condensed nucleus) and late apoptotic cells (red condensed nucleus), which are considered to be basic apoptotic cells. After adding ASA (15mM), the late apoptotic cells increased significantly, and the early apoptotic cells increased slightly. After adding PZ (70μM), the number of apoptotic cells increased by ASA decreased significantly, while PZ itself did not affect cell viability, and there were no necrotic cells in the two groups.

  2.4 The influence of PZ and ASA on the expression of HSP70

  "Unstimulated RIE1 cells express a small amount of HSP70. After ASA (15mM), PZ (70μM) and ASA and PZ pretreatment (Figure 4A and B), the expression of HSP70 in the cells was only significantly reduced after ASA, and was time-dependent. However, PZ significantly restored the expression of HSP70 at 15, 24, and 39 h. When PZ pretreatment was used alone, the expression of HSP70 in RIE1 cells increased in a time-dependent manner.

  2.5 Screening of HSP70 specific siRNA and confirmation of HSP70 silencing after transfection with RIE1

  To further study the role of PZ-induced HSP70 in ASA-induced apoptosis of RIE1 cells, we silenced HSP70 in RIE1 with siRNA. First tried three HSP70 siRNAs, of which only one siRNA (rat hspa1b-2868) can completely silence HSP70. Therefore, the most effective HSP70 siRNA was selected for all subsequence RIE1 transfection experiments. The time for HSP70 siRNA to silence HSP70 expression after transfection was also evaluated, and it was found that 8h after transfection, the cells could not express HSP70 within 39h after 30 min of heat shock at 43℃. This heat shock stimulation method significantly induced the expression of HSP70 in naïve RIE1 and Mock-RIE1 cells (negative control) within 24 hours.

  Figure 5 Screening of HSP70 specific siRNA and confirmation of HSP70 silencing after transfection with RIE1

  2.6 PZ cannot induce HSP70 to silence HSP70 expression in RIE1

  "39h after administration, the expression of HSP70 in naïve RIE1 and Mock-RIE1 increased significantly. However, PZ could not induce the expression of HSP70 in HSP70 silenced RIE1.

  In order to further confirm the effectiveness of HSP70 silencing, the expression of HSP70 after ASA and ASA combined with PZ pretreatment was studied. The results showed that unstimulated naïve RIE1 and Mock-RIE1 cells expressed a small amount of HSP70, and after PZ pretreatment, the expression of HSP70 was significantly increased. However, after treating these cells with ASA (15mM) for 15h, HSP70 was significantly reduced. In contrast, unstimulated HSP silenced RIE1 did not express HSP70, nor did PZ or ASA increase HSP70 expression in these cells.

  2.7 HSP70 silencing leads to loss of PZ protection against ASA-induced apoptosis

  ASA significantly induces apoptosis in naïve RIE1 and Mock-RIE1, while cells pretreated with PZ can significantly inhibit apoptosis. However, the silencing of HSP70 in RIE1 cells resulted in a significant loss of the protective effect of PZ on ASA-induced apoptosis, indicating that HSP70 plays a vital role in this process.

  2.8 PZ inhibits ASA-induced late apoptosis

  Perform Annexin V-FITC binding analysis and PI staining to determine the type of apoptosis induced by ASA. In each experiment, PI negative/annexin V negative cells represent live cells, PI positive/annexin V negative cells represent necrotic cells, PI negative/annexin V positive cells represent early apoptotic cells, and PI positive/annexin V positive cells represent late stages Apoptotic cells. As shown in Figure 8, PZ mainly inhibits ASA-induced late apoptosis in a HSP70-dependent manner.

  Discuss

  The results showed that non-steroidal anti-inflammatory drugs (acetylsalicylic acid, ASA) induced apoptosis of small intestinal epithelial cells RIE1 in a dose-dependent manner, and polyprezinc significantly reduced the ASA-induced cytotoxicity in RIE1 cells by promoting the expression of HSP70. It has a significant protective effect on cells. What is important is that polyprezinc does not affect cell viability by itself, indicating that the drug is safe.

  Ruilaisheng (Polyprezinc Granules) is not only a safe and effective gastric mucosal protective agent, but also can be widely used in the clinical treatment of intestinal mucosal damage caused by non-steroidal anti-inflammatory drugs (SAS).

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