Application of mitochondrial OS in Stat3 activation and liver inflammation generation

Abstract

The invention discloses application of mitochondrial OS in Stat3 activation and liver inflammation generation, and proves that continuous replication of HBV in liver cells can cause mitochondrial respiratory dysfunction and further cause ROS accumulation. As the ROS level is increased, Nrf-2 protein expression is increased, and transcription up-regulation of cell factors IL-6 and IL-8 is caused. The IL-6 and the IL-8 are self-secreted / paracrine to be combined with membrane receptors of the IL-6 and the IL-8, and Janus kinase 2 (Jak2) is collected and activated. The Stat3 is then recruited, and then phosphorylated by Jak2. After phosphorylation, the p-Stat3 is subjected to homodimerization, and translocation is allowed to enter a cell nucleus, so that transcriptional activation of a group of inflammatory genes, such as Saa1, S100a9 or Icam1 and the like, is promoted. The sharp increase of the inflammatory mediators causes the sharp increase of infiltration of intrahepatic immune and inflammatory cells (neutrophil (N), mononuclear cells (Mo) or macrophages (Mphi) and the like), and further causes the occurrence of liver inflammation. The active oxygen scavenger N-NAC can reduce generation of active oxygen and inhibit liver inflammatory response caused by the active oxygen.

Description

Technical Field

[0001] The present invention relates to the function and application field of genes, and specifically discloses the application of mitochondrial OS in Stat3 activation and liver inflammation. Background Art

[0002] In recent years, hepatitis B virus (HBV) infection remains a major public health problem. In immunocompetent adults, most HBV infections resolve spontaneously, with less than 5% becoming chronic infections, while up to 90% of chronic HBV infections are acquired during infancy and early childhood. The natural history of chronic hepatitis B virus infection (CHB) includes four distinct phases, each characterized by different levels of viral replication, viral antigen expression, and liver inflammation. The first phase, called the “immune tolerance (IT) phase,” is usually observed after infection acquired during the perinatal or early childhood period and is characterized by high viral replication, the presence of HBeAg, normal serum alanine aminotransferase (ALT) levels, and minimal or no liver inflammation or fibrosis on biopsy. During adolescence or adulthood, these patients in the IT phase typically enter the second phase, the “immune active (IA) phase,” characterized by high fluctuating ALT levels, declining HBV DNA and HBeAg levels, and moderate to severe liver inflammation or fibrosis. However, the mechanisms that trigger the transition from the IT phase to the IA phase remain unclear.

[0003] In previous studies, this mechanism was explored by dynamically analyzing the liver transcriptome of a HBV transgenic mouse (HBV-Tg) model with spontaneous liver inflammation. The results showed that activation of signal transducer and activator of transcription 3 (Stat3) triggered the expression of inflammation-related genes and controlled the early stages of HBV-induced liver inflammation. These findings provide clues for further investigation of the activation mechanism of upstream regulators of Stat3. Interestingly, some liver transcriptome-based studies have found that many metabolic pathways, such as oxidative phosphorylation, fatty acid metabolism, and bile acid metabolism, are downregulated in CHB patients with relatively high immune gene expression and immune cell infiltration. It is well known that mitochondria are the main sites of oxidative phosphorylation and ROS production, and are also central hubs for regulating metabolic pathways and controlling cell signaling. Therefore, these evidences suggest that mitochondrial dysfunction in chronic HBV infection may play an important role in these metabolic changes and inflammatory infiltration. Recent studies have shown that overexpression of HBV X protein (HBx) in experimental models can induce changes in mitochondrial structure and function and cause oxidative stress (OS). In addition, mitochondrial OS, mtDNA damage, and related changes in mitochondrial function and dynamics have been observed in CHB patients with advanced fibrosis or cirrhosis. However, whether mitochondrial OS serves as a key initiator of HBV-induced inflammation and contributes to the transition from IT stage to IA stage has not been found.

[0004] Therefore, this paper will focus on the changes in mitochondrial OXPHOS and ROS production induced by HBV replication, as well as the role of mitochondrial OS in Stat3 activation and the occurrence of liver inflammation, bringing some new insights into the complex molecular mechanisms of the transition from IT stage to IA stage of chronic HBV infection. Summary of the invention

[0005] In view of the above analysis, the present invention aims to further discover the molecular mechanism of Stat3 activation. The present invention is achieved by the following technical means:

[0006] The application of mitochondrial OS in Stat3 activation and liver inflammation. The present invention confirms that the continuous replication of HBV in hepatocytes can cause mitochondrial respiratory dysfunction and further lead to ROS accumulation. As ROS levels increase, Nrf-2 protein expression increases, leading to upregulation of cytokine IL-6 and IL-8 transcription. Autocrine / paracrine IL-6 and IL-8 bind to their membrane receptors, recruiting and activating Janus kinase 2 (Jak2). Stat3 is then recruited and subsequently phosphorylated by Jak2. After phosphorylation, p-Stat3 undergoes homodimerization, allowing translocation into the cell nucleus, thereby promoting the transcriptional activation of a group of inflammatory genes, such as Saa1, S100a9 or Icam1. The surge of these inflammatory mediators causes intrahepatic immune and inflammatory cells [neutrophils (N), monocytes (Mo) or macrophages The infiltration of liver cells increases sharply, which in turn triggers the occurrence of liver inflammation. The reactive oxygen scavenger N-NAC can reduce the production of reactive oxygen species and inhibit the liver inflammation response caused by reactive oxygen species.

[0007] The beneficial effects of the present invention are:

[0008] The present invention found that the activation of signal transducer and activator of transcription 3 (Stat3) triggered the expression of inflammation-related genes and controlled the early stages of HBV-induced liver inflammation. At the same time, the excessive production of ROS during chronic HBV infection also played a key role in triggering the inflammatory response, providing a new perspective for a deeper understanding of the mechanism of inflammation during persistent viral infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are briefly introduced below.

[0010] Figure 1 These are PAAV / HBV1.2 mouse model-related markers; among them: Figure 1A shows the dynamic changes of HBVDNA, HBsAg, and HBeAg levels in the peripheral blood of pAAV / HBV1.2 mice; Figure 1 B shows the dynamic changes of serum ALT and liver inflammation levels in pAAV / HBV1.2 mice.

[0011] Figure 2 The ROS-Stat3 pathway mediates early inflammatory activation in the liver tissue of PAAV / HBV1.2 mice; Figure 2 A shows the dynamic changes of ROS levels in the liver of pAAV / HBV1.2 mice; Figure 2 B shows the dynamic changes of oxidative stress levels in the liver of pAAV / HBV1.2 mice; Figure 2 C is the protein related to the early activation of Stat3 pathway in the liver of pAAV / HBV1.2 mice; Figure 2 D is the mRNA expression of genes related to early Stat3 pathway activation in the liver of pAAV / HBV1.2 mice.

[0012] Figure 3 To clear ROS in the body, it can downregulate the activation of the JAK2 / Stat3 pathway and thus inhibit liver inflammation; among them: Figure 3 A shows the dynamic levels of ROS and oxidative stress in the liver of pAAV / HBV1.2 mice after injection of the ROS scavenger NAC; Figure 3 B is HE staining of the liver of pAAV / HBV1.2 mice after injection of ROS scavenger NAC; Figure 3 C is the level of inflammation in the liver of pAAV / HBV1.2 mice after injection of ROS scavenger NAC; Figure 3 D shows the Nrf2 protein level and Jak / Stat3 pathway protein expression level in the liver of pAAV / HBV1.2 mice after injection of ROS scavenger NAC. DETAILED DESCRIPTION

[0013] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0014] Mouse sample collection. Blood was collected from the eyeballs of pAAV / HBV1.2 and WT mice, anticoagulated, and centrifuged to obtain plasma for HBVDNA, HBV markers, and liver function tests; the lower blood cells were used for immune cell subset detection. After blood collection, the mice were sacrificed by cervical dislocation, and the liver tissue was dissected and washed with saline and weighed. Part of the liver tissue was fixed with 4% paraformaldehyde for pathological examination. An appropriate amount of liver tissue was quickly stored in liquid nitrogen for subsequent gene expression RNA and protein detection, and the remaining liver tissue was used to extract intrahepatic immune cells and flow cytometry.

[0015] Example 1

[0016] The serum HBV DNA, HBeAg, ALT and AST levels of pAAV / HBV1.2 mice and wild-type mice at 2, 3, 4 and 5 weeks of age were measured using a fully automatic biochemical instrument.

[0017] The results are as follows Figure 1 As shown in A, in the comparison between pAAV / HBV1.2 mice and wild-type mice of the same age, HBV virus-related markers in pAAV / HBV1.2 mice were all positive, indicating a successful model.

[0018] Example 2

[0019] Hematoxylin and eosin (HE) staining of liver tissue

[0020] 1. Use distilled water to dilute anhydrous ethanol to 100%, 95%, 85%, and 75% ethanol;

[0021] 2. Take a liver tissue specimen of appropriate size preserved in 4% paraformaldehyde, embed it in paraffin into a regular cube, use a pathological slicer to cut 6-8um thick tissue sections, adsorb them on adhesive slides, and use an immunohistochemistry pen to draw lines along the edge of the tissue;

[0022] 3. Immerse in xylene to dewax for 10 minutes, repeat once;

[0023] 4. Dehydrate by immersion in a series of ethanol (100%, 95%, 85%, 75%), with each gradient dehydration lasting 3 minutes;

[0024] 5. Immerse in distilled water for 2 minutes;

[0025] 6. Add hematoxylin solution and dye for 8 minutes, then wash off the floating color under running distilled water;

[0026] 7. Add differentiation solution and differentiate for 40 seconds, then rinse under running tap water for 6 minutes;

[0027] 8. Immerse in eosin stain for 1 minute and pour off the excess stain;

[0028] 9. Immerse in 75% ethanol, 85% ethanol, 95% ethanol and 100% ethanol①, immerse for 2-3 seconds in each gradient;

[0029] 10. Wash with 100% ethanol② for 1 min, immerse in xylene① and xylene② for 1 min each, and seal with neutral gum;

[0030] 11. After the neutral gum is completely dry, scan it with a digital slice analysis scanner and analyze the inflammatory infiltration in the liver with reference to the Ishak scoring system.

[0031] The results are as follows Figure 1 As shown in B, compared with the control group mice, there was no or very little immune cell infiltration in the liver tissue of pAAV / HBV1.2 mice at 2 and 3 weeks of modeling; at 4 and 5 weeks, obvious and large amounts of immune cell infiltration appeared in the portal area of ​​the liver.

[0032] Example 3

[0033] Hematoxylin and eosin (HE) staining of liver tissue

[0034] Flow cytometry

[0035] 1. Prepare several EP tubes and number them for use;

[0036] 2. Add 50ul liver tissue immune cell suspension into the tube, then add 0.5ul FC blocking solution and react at room temperature in the dark for 10 minutes;

[0037] 3. Mix the required volume of flow cytometry antibody in advance as needed, add it to the corresponding EP tube, and incubate at 4°C in the dark for 30 minutes;

[0038] 4. After incubation, add 1 ml of PBS solution to each tube, mix by inverting, and centrifuge at 4°C, 2000 rpm / min for 10 min;

[0039] 5. After discarding the supernatant, add 200ul FACS solution to resuspend the cells and analyze them by flow cytometry;

[0040] 6. Use Flowjo software for data analysis.

[0041] The results are as follows Figure 1 As shown in B, compared with the control group mice, the number of various immune cells in the liver of mice increased at 4 weeks after modeling.

[0042] Example 3: Oxidative stress index detection

[0043] (1) Detection of reduced glutathione (GSH) content

[0044] 1. Weigh 0.1g liver tissue, transfer to a pre-cooled homogenizer, add 1mL of reagent, homogenize several times, centrifuge at 4℃, 8000g for 10min, take the supernatant and place it at 4℃ for testing;

[0045] 2. Preheat the microplate reader for more than 30 minutes and adjust the wavelength to 412nm;

[0046] 3. Prepare GSH standard solution according to the table below;

[0047]

[0048] 4. Take the sample to be tested for testing. The sample addition method is as follows (96-well plate sample addition)

[0049]

[0050] 5. After mixing, let stand at room temperature for 2 minutes, measure the absorbance of the assay tube, standard tube and blank tube at 412nm, record them as A assay, A standard and A blank, calculate ΔA = A assay - A blank, ΔA standard = A standard - A blank. According to the concentration of the standard tube (x, μg / ml) and the absorbance ΔA standard (y, ΔA standard), establish a standard curve. According to the standard curve, substitute ΔA (y, ΔA) into the formula to calculate the sample concentration (x, μg / ml);

[0051] 6. GSH content (μg / g mass) = x × V sample ÷ (V sample ÷ V sample total × W) = x ÷ W, V sample total: total volume of supernatant, 1 ml; V sample: volume of supernatant added to the reaction system, 20 μL = 0.02 ml; W: sample mass, 0.1 g.

[0052] (2) Detection of oxidized glutathione (GSSG) content

[0053] 1. Weigh 0.1g liver tissue, transfer to a pre-cooled homogenizer, add 1mL of reagent, homogenize several times, centrifuge at 4℃, 8000g for 10min, take the supernatant and place it at 4℃ for testing;

[0054] 2. Preheat the microplate reader for more than 30 minutes, adjust the wavelength to 412nm, and place reagent 3 in a 37℃ water bath for 30 minutes; prepare the GSSG standard solution according to the table below;

[0055]

[0056]

[0057] 3. Take the sample to be tested for testing. The sample addition method is as follows (96-well plate sample addition)

[0058]

[0059] 4. Start timing when adding reagent VI, mix quickly, measure the light absorption at 412 nm for 30s and 150s A1 measurement (A1 standard, A1 blank) and A2 measurement (A2 standard, A2 blank), and calculate ΔA measurement = A2 measurement - A1 measurement, ΔA standard = A2 standard - A1 standard, ΔA blank = A2 blank - A1 blank;

[0060] 5. Draw a standard curve with the concentration of the standard tube (μg / ml) as the abscissa x and (ΔA standard - ΔA blank) as the ordinate y, and substitute (ΔA determination - ΔA blank) into the formula to calculate the sample concentration (μg / ml);

[0061] 6.GSSG (μg / g mass) = x × V sample ÷ (V sample ÷ V sample total × W) = x ÷ W, V sample total: total volume of supernatant, 1 mL; V sample: volume of supernatant added to the reaction system, 20 μl = 0.02 ml; W: sample mass, 0.1 g.

[0062] (3) Detection of reactive oxygen species (ROS) content in tissues

[0063] 1. Take liver tissue of appropriate size and put it into EP tube, freeze it in liquid nitrogen for about 10 seconds, and store it in -80℃ ultra-low temperature refrigerator for later use;

[0064] 2. After taking out the tissue, place it in a mold, add OCT embedding agent to embed the tissue into a regular cube, cut 6-8μm thick tissue sections at -20℃ using a freezing slicer, adsorb them on an adhesive slide, and store them in a -20℃ refrigerator for testing;

[0065] 3. Take out the slices and rewarm them at room temperature for 10 minutes. Use PBS to wash the excess OCT on the slices and absorb the residual liquid with clean filter paper.

[0066] 4. Use an immunohistochemistry pen to draw a circle along the edge of the tissue section, add DCFH-DA working solution, and incubate at 37°C in the dark for 30 minutes;

[0067] 5. After the incubation, take out the slices, discard the excess DCFH-DA working solution, place them in TBST solution, wash on a shaker for 5 minutes, repeat 3 times, and use clean filter paper to absorb the residual liquid;

[0068] 6. Add Hoechst staining solution and incubate at room temperature in the dark for 5 minutes;

[0069] 7. After incubation, remove the slices, discard the excess Hoechst working solution, place them in TBST solution, wash on a shaker for 5 minutes, and use clean filter paper to absorb the remaining liquid;

[0070] 8. Add anti-fluorescence attenuation mounting agent, cover the slide with a coverslip, and photograph with a confocal microscope;

[0071] 9. Use ImageJ software to analyze fluorescence intensity.

[0072] result Figure 2 As shown in A and B, at 2 weeks after modeling, the ROS levels in the liver of pAAV / HBV1.2 mice and control mice were similar; at 3, 4, and 5 weeks, the ROS fluorescence intensity of pAAV / HBV1.2 mice was significantly higher than that of control mice; at 3 weeks after modeling, oxidative stress in pAAV / HBV1.2 mice gradually became obvious, and ROS in the liver began to increase significantly. At 4 and 5 weeks, oxidative stress in pAAV / HBV1.2 mice was further aggravated.

[0073] Implementation Example 4

[0074] Western Blot detection of Stat3, p-Stat3, Jak2, p-Jak2, Nrf2 and β-actin protein expressions in liver tissue

[0075] (1) Tissue protein extraction

[0076] 1. Take about 30 mg of frozen liver tissue into an EP tube and add 500 μl of protein lysis buffer;

[0077] 2. 6500 rpm / min, oscillate for 23 s, rest for 10 s, repeat once to make tissue homogenate;

[0078] 3. Place on ice for 30 minutes, and shake and mix manually every 5 minutes to promote full reaction;

[0079] Centrifuge at 4.4°C, 12,000 g for 5 min, aspirate the supernatant (protein extract), and place on ice to measure the concentration;

[0080] The protein concentration was determined by BCA protein kit as in Example 2

[0081] Western Blot gel electrophoresis is the same as in Example 2

[0082] Detection of Stat3 gene regulation in liver tissue by qPCR

[0083] (1) Extract RNA from liver tissue using a kit

[0084] 1. Grind about 30 mg of liver tissue quickly in liquid nitrogen, transfer to a grinding tube, immediately add 500 ul TRIzol and grinding beads, mix well, and leave at room temperature for 5 minutes to ensure sufficient reaction;

[0085] 2. 6500 rpm, oscillate for 23 s, rest for 10 s, repeat once to make tissue homogenate;

[0086] Centrifuge the homogenate at 10,000 g for 10 min at 3.4°C.

[0087] 4. Take another clean EP tube and place it on ice, add 250ul of anhydrous ethanol, and take an equal amount of supernatant from step 3 and shake it thoroughly;

[0088] 5. Transfer all the mixed liquid into a clean adsorption column, put a collection tube on the lower end, and centrifuge at 4°C, 10,000 g for 30 seconds;

[0089] 6. Discard the collection tube and the liquid in it and replace it with a new collection tube;

[0090] 7. Add 400ul Direct-zol RNAprewash buffer to the adsorption column, centrifuge at 10000g for 30s at 4℃, discard the liquid in the collection tube, and repeat once;

[0091] 8. Add 700ul RNAwash buffer to the adsorption column, centrifuge at 4℃, 10000g for 2min, discard the collection tube and the liquid in it, and put a clean EP tube on the lower end;

[0092] 9. Add 50ul DNase / RNase-free water to the adsorption column, centrifuge at 10000g for 30s at 4℃, and collect RNA in the EP tube;

[0093] (2) RNA concentration determination is the same as in Example 2

[0094] (3) RNA reverse transcription into cDNA is the same as in Example 2

[0095] (4) Reverse Transcription PCR amplification experiment is the same as in Example 2.

[0096] Elisa detection of IL-6 and IL-8 levels in mouse peripheral blood (the steps are the same for both)

[0097] 1. Take out the reagent kit and equilibrate at room temperature for 30 minutes;

[0098] 2. Prepare washing buffer, biotinylated antibody working solution, and enzyme conjugate working solution as needed;

[0099] 3. Prepare the standard product according to the table below and set aside;

[0100]

[0101] 4. Choose the appropriate number of strips according to the number of samples to be tested, and add 1 well as a blank control. Add 100 μl of the corresponding serum to be tested and different concentrations of standard, add 100 μl of standard diluent to the blank well, cover the reaction plate with sealing paper, and incubate at 37°C in the dark for 90 minutes;

[0102] 5. After the incubation, add 350 μl of washing buffer to each reaction well, let it stand for 30 seconds and then discard it. Repeat 4 times. After the last wash, pat the remaining liquid in the well dry on a clean filter paper.

[0103] 6. Add 100 μl of biotinylated antibody working solution to each well (except blank wells), cover the reaction plate with sealing paper, and incubate at 37°C in the dark for 60 minutes;

[0104] 7. After the incubation, add 350 μl of washing buffer to each reaction well, let it stand for 30 seconds and then discard it. Repeat 4 times. After the last wash, pat the remaining liquid in the well dry on a clean filter paper.

[0105] 8. Add 100 μl of enzyme conjugate working solution to each well (except blank wells), cover the reaction plate with sealing paper, and incubate at 37°C in the dark for 30 minutes;

[0106] 9. After the incubation, add 350 μl of washing buffer to each reaction well, let it stand for 30 seconds and then discard it. Repeat 4 times. After the last wash, pat the remaining liquid in the well dry on a clean filter paper.

[0107] 10. Add 100 μl of color developer to each well, cover the reaction plate with sealing paper, and incubate at 37°C in the dark for 15 min;

[0108] 11. Add 100 μl of stop solution to each well, mix well, and immediately measure the OD value at 450 nm.

[0109] The results are as follows Figure 2 As shown in C and D, Western blot and qPCR results showed that chronic HBV replication could lead to increased ROS, which mediated the occurrence of liver inflammation in HBV-replicated mice through the Nrf2-IL6 / IL-8-Jak2 / Stat3 pathway.

[0110] Implementation Example 5 is the same as Implementation Example 3

[0111] The results are as follows Figure 3 As shown in A, NAC treatment can significantly reduce the levels of ROS and oxidative stress in the liver of pAAV / HBV1.2 mice.

[0112] Implementation Example 6 is the same as Implementation Example 2

[0113] The results are as follows Figure 3As shown in B and C, reducing ROS can inhibit inflammatory infiltration in the liver of pAAV / HBV1.2 mice.

[0114] Implementation Example 7 is the same as Implementation Example 4

[0115] The results are as follows Figure 3 As shown in D, reducing ROS significantly downregulated the Nrf2 protein level and Jak2 / Stat3 pathway activation in the liver of pAAV / HBV1.2 mice.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of mitochondrial OS in Stat3 activation and liver inflammation.

2. The use according to claim 1, wherein excessively elevated ROS in hepatocytes enhances the activation of the Jak2 / Stat3 pathway via the Nrf2-IL-6 / IL-8 pathway and thereby upregulates the expression of acute inflammation-related factors.

3. The use according to claim 1, wherein the removal of ROS in vivo can downregulate the activation of the Jak2 / Stat3 pathway and thus inhibit the occurrence of liver inflammation.

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