Study on the effect of Mongolian Medicine Qiwei Qinggan 1 Powder on Hepatic Fibrosis through JAK2/STAT3 Pathway 2

6 Background: To study the anti-hepatic fibrosis effect and explore the mechanism of 7 Qiwei Qinggan Powder (QGS-7) in vivo and in vitro. 8 Methods: Carbon tetrachloride (CCl 4 )-treated rats and hepatic stellate cells (HSCs) 9 were used. Alanine Aminotransferase (ALT), Aspartate transaminase (AST) and 10 Alkaline Phosphatase (ALP) were detected in serum of rats in each group, 11 hydroxyproline (HYP) was detected in liver tissue. Formalin-fixed liver specimens 12 were stained with hematoxylin and eosin (H&E) reagent, Masson trichrome, and then 13 analyzed. The expression of Alpha smooth muscle actin (α-SMA) in liver was 14 detected by immunohistochemistry. The expression level of Collagen I, α-SMA, Janus 15 kinase 2 (JAK2), and signal transducer and activator of transcription 3(STAT3) 16 mRNA were determined by real Time polymerase chain reaction (RT-qPCR). Meanwhile, the protein expression levels of α-SMA, Collagen I, JAK2, 23 phosphorylation-JAK2 (p-JAK2), STAT3 and phosphorylation-STAT3 (p-STAT3) 24 were determined by Western Blot. The proliferation of HSC was detected by MTT 25 and the apoptosis was detected by flow cytometry. 26 Results: QGS-7 treatment significantly improved the liver function of rats as 27 indicated by decreased serum enzymatic activities of ALT, AST and ALP. Meanwhile, 28 the HYP of liver was significantly decreased. Histopathological results indicated that 29 QGS-7 alleviated liver damage and reduced the formation of fibrosis septa. Moreover, 30 QGS-7 significantly attenuated expressions of α-SMA, Collagen I, JAK2, p-JAK2, 31 STAT3, p-STAT3 relative mRNA and protein level in the rat hepatic fibrosis model 32 and HSCs. And QGS-7 can inhibit HSCs proliferation and promote it apoptosis. Conclusion: Mongolian medicine the effect of treating hepatic and can inhibit the activation, proliferation and promote apoptosis of

Meanwhile, the protein expression levels of α-SMA, Collagen I, JAK2, 23 phosphorylation-JAK2 (p-JAK2), STAT3 and phosphorylation-STAT3 (p-STAT3) 24 were determined by Western Blot. The proliferation of HSC was detected by MTT 25 and the apoptosis was detected by flow cytometry. which can be used to treat various liver diseases [7]. It has been shown that QGS-7 57 has a good therapeutic effect on acute liver injury [8][9]. However, its therapeutic 58 effect and mechanism on hepatic fibrosis have not been reported. 59 We calculated the liver index of rats, measured the contents of ALT, AST and 60 ALP in serum, HYP in liver, observed the liver injury by HE staining, and counted the 61 collagen content by Masson staining. It was confirmed that QGS-7 has a good effect 62 on repairing liver injury and alleviating hepatic fibrosis. In addition, we also used 63 immunohistochemistry to detect α-SMA in liver tissue, RT-qPCR and Western blot to  In order to study the mechanism of QGS-7 in the treatment of hepatic fibrosis, 67 we extracted RNA from the liver tissue of each group and analyzed the transcriptome 68 sequence. Using bioinformatics, we screened out the signaling pathways with prescription to intervene hepatic fibrosis through JAK2/STAT3 signaling pathway. We 76 hope to provide experimental data for the effect, target and mechanism of QGS-7 in 77 the treatment of hepatic fibrosis.

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The Minimum Standards of Reporting Checklist contains details of the experimental 80 design, and resources used in this study (Additional file 1). anesthesia. Blood was obtained from the abdominal aorta, and the liver was excised.

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The liver was immediately frozen for biochemical measurements or fixed in formalin 107 for histochemical examination. 109 Wistar rats were randomly divided into two groups (8 rats per group): Control group 110 6 and QGS-7 groups. QGS-7 was given once a day according to 10 times of the lowest 111 adult dose [1350 mg/(kg · d)]. On the 7th day, the rats fasted 12 h before gavage and 112 carried out the experiment within 1-2 h after gavage. The blood was collected from 113 abdominal aorta, and then placed for 20 minutes, centrifuged in a centrifuge (4 °C , 114 3000 r/min, 15 min). After centrifugation, the serum was filtered with 0.22 μM filter 115 membrane, which was called drug serum. In the control group, the drug was replaced 116 by normal saline, and the preparation method was the same as before. After being 117 inactivated at 56 °C for 30 minutes, the drug serum was stored in a refrigerator at -     Screening signal pathway by transcriptome sequencing 145 The first step is to extract RNA from liver tissue and evaluate its quality, then purify,   China) was used for reverse transcription. Then the relative mRNA expression of 157 α-SMA, collagen I, JAK2 and STAT3 in rat liver and HSC was detected by RT-qPCR.

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The data were processed by 2 -ΔΔCt method. The primers (Shanghai Sengon Biological 159 and Technological Company, Shanghai, China) for each gene are shown in the Table   160 1.

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Western blot analysis 162 The protein in rat liver and HSC was extracted respectively. After quantitative 163 analysis by BCA method, the sample buffer was added. After protein boiling, the  180 The logarithmic phase cells were collected and seeded in 6 well plates. Each group 181 was set with 3 duplications and cultured for 12 hours. Discard the original culture 182 medium, add the corresponding concentration of serum culture medium for 24 hours.   188 All results were presented as mean ± standard deviation (SD). Statistical analysis was 189 performed with SPSS software (version 24). The statistical significance between 190 groups was analyzed using one way ANOVA. The difference was considered 191 significant at P < 0.05. 194 In order to study the anti-fibrosis effect of QGS-7, we first studied the therapeutic showed that in the blank group, the structure of liver lobule was complete, the 217 hepatocytes were arranged orderly and the plasma was even. In the model group, the 218 structure of liver lobule was destroyed, the arrangement of liver plate was disordered,

In vitro experiments verify that QGS-7 has anti fibrosis effect through JAK2/STAT3
264 13 signaling pathway 265 The results of RT-qPCR showed that compared with the control group, the relative 266 mRNA expression of α-SMA and collagen I in the low and high dose serum group 267 decreased significantly, and the relative mRNA expression of α-SMA and collagen I 268 in the middle dose serum group decreased significantly ( Figure 4A).

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Western blot results showed that compared with the control group, the expression 270 level of α-SMA and Collagen I protein in the low, middle and high dose serum groups 271 decreased significantly ( Figure 4B).

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The results of RT-qPCR showed that compared with the control group, the were 21.36%, 33.26% and 33.00% (Table 3).

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FITC labeled Annexin and PI double staining can be used to distinguish three 290 types of cells: living cells, early apoptotic cells and late apoptotic and necrotic cells.

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The results showed that compared with the control group, the apoptosis rate of the 292 low dose serum group was significantly higher; the apoptosis rate of the high dose 293 group was significantly higher ( Figure 4E).

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Fibrosis is a process closely related to organ damage, which plays a role in preventing