| Objective:1.Summarizing the theory and drug characteristics of traditional Chinese med-icine in the treatment of hepatic fibrosis with liver stagnation and spleen deficiency by lit-erature analysis.2.Observing the clinical efficacy of HXRGD in the treatment of patients with liver stagnation and spleen deficiency type of chronic hepatitis B（CHB）hepatic fi-brosis.3.Researching the effect of HXRGD on NOX4/ROS pathway and ferroptosis markers in rats with hepatic fibrosis in vivo.4.Researching whether HXRGD can promote HSC ferroptosis through NOX4/ROS pathway and play a role in anti-hepatic fibrosis in vitro..Method:1.China National Knowledge Infrastructure,Wanfang Database,Weipu Data-base,Pubmed,Web of Science to the‘Soothing the liver and strengthening the spleen’or‘liver depression or spleen deficiency’or‘harmonizing the liver and spleen’and‘hepatic fibrosis’as keywords to carry on the literature search,duplication,conference papers,no record of prescriptions,experience,study on Traditional Chinese Medicine syndrome type,to extract the literature of Chinese medicine.All the prescriptions were screened,and the same prescriptions were recorded only once.The herbal names were standardized accord-ing to the Chinese Pharmacopoeia 2020 edition and Chinese Materia Medica.The taste and property of herbal was analyzed,and complex network analysis and association rule analysis were conducted.2.A total of 48 liver stagnation and spleen deficiency type CHB patients with hepatic fi-brosis diagnosed by liver biopsy or Fibro Touch at the Department of Hepatology,Affiliat-ed Hospital of Traditional Chinese Medicine,Xinjiang Medical University from January2022 to August 2023 were included.They were randomly divided into treatment group（n=23）and control group（n=25）.The treatment group was treated with HXRGD and anti-viral therapy with nucleoside drugs,while the control group was treated with antiviral therapy with nucleoside drugs alone.The course of treatment in both groups was 24 weeks.General data,laboratory examination,liver stiffness measurements（LSM）,TCM syndrome scores,calculating fibrosis 4 score（FIB-4）,aspartate aminotransferase to platelet ratio in-dex（APRI）of patients in the two groups were collected before and after treatment,and statistical analysis was conducted to compare the degree of liver fibrosis in the treatment group and the control group before and after treatment,and to evaluate the clinical effica-cy of HXRGD in the treatment of hepatic fibrosis.3.Sixty SD rats were divided into six groups of 10 rats each using the random number ta-ble method.The subgroups were as follows:（1）normal control group,（2）model control group,（3）colchicine group,（4）HXRGD low-dose group,（5）HXRGD medium-dose group,and（6）HXRGD high-dose group.Excepting the normal control group,the other five groups were treated with carbon tetrachloride to establish the rat model of hepatic fi-brosis.At the 8th week of modeling,the rat model of hepatic fibrosis was evaluated.The normal control group and model group were intragastric with distilled water,the colchi-cine group was intragastric with colchicine at a dose of 0.02 mg/m L,and the HXRGD low,medium and high dose groups were intragastric with doses of 0.927 g/m L,1.854 g/m L and3.708 g/m L,respectively,with an intragastric volume of 10 m L/kg for 4 consecutive weeks.HE staining and Masson staining were used to observe the degree of liver inflam-mation and fibrosis,and Ishak score was performed.The levels of GSH,GSSG,MDA,Fe and Fe2+in liver tissue were detected by kits.DCFH-DA probe was used to detect the ROS level in rat liver tissue.Western blot and RT-q PCR were used to detect the expression of NOX4 protein and NOX4 m RNA in rat liver tissue,and statistical analysis was per-formed.4.A total of 18 SD rats were divided into normal control group and HXRGD group by random number table method.The HXRGD group was given intragastric administration of HXRGD at a concentration of 3.708 g/m L,while the normal control group was given in-tragastric administration of distilled water with an intragastric volume of 10 m L/kg,once a day for 6 consecutive days,and the serum was taken for reserve use on the 7th day.HSC-T6 cells were divided into 7 groups,which were（1）blank control group,（2）normal rat serum group,（3）TGF-β1+normal rat serum group,（4）TGF-β1+erastin group,（5）TGF-β1+HXRGD low-dose medicated serum group,（6）TGF-β1+HXRGD medium-dose medi-cated serum group,and（7）TGF-β1+HXRGD high-dose medicated serum group.After group intervention,CCK8 and Annexin V-APC/7-AAD assays were used to detect cell proliferation and apoptosis,respectively.Kits were used to detect GSH,GSSG,Fe,and MDA levels in HSC-T6 cells,respectively,and DCFH-DA probes were used to detect ROS levels in the cells.Western blot and RT-q PCR assays were used to detect cellular NOX4 protein Western blot and RT-q PCR were used to detect cellular NOX4 protein and NOX4 m RNA expression respectively.BODIPY 581/591 C11 probe was used to detect cellular lipid ROS levels and observed by laser confocal microscopy and transmission electron microscopy was used to observe mitochondrial morphology in HSC-T6 cells.Results:1.181 prescriptions were finally extracted from 181 papers,a total of 193 Chi-nese herbals were obtained,and the top 10 were:Chai hu（141）、Bai zhu（123）、Bai shao（108）、Fu ling（102）、Gan cao（96）、Huang qi（96）、Dan shen（92）、Bie jia（68）、Dang shen（62）、Dang gui（61）.The drugs of cold and heat were the most and the least among the Chinese herbals of taste.Bitter drugs were the most common,followed by sweetish Chinese herbals.The liver channel,spleen channel and stomach channel were the most channel tropism.In the complex network analysis,41 srong association rule,65 me-dium association rule and 2912 weak association rule were found,and the core herbal pairs were Chai hu and Bai zhu.The results of association rule analysis showed 105 asso-ciation drug combinations,including 14 association analysis results of two herbal pairs and 91 association analysis results of three herbal pairs.According to the clustering analy-sis of the top 30 Chinese herbals,they can be divided into 3 categories:the first is disperse the liver and rectify qi with invigorating qi and invigorating spleen and dampness.The second is manage qi and activating blood dissipating stasis with removing blood stasis and dispersing phlegm dispersing stasis.The third is clearing heat and promoting diuresis with promoting qi and dispersing blood stasis.2.A total of 48 patients were included,including 28 males and 20 females,with an aver-age age of 49.77±9.71.There were 23 cases in treatment group and 25 cases in control group.There was no significant difference in baseline data between the two groups（P>0.05）,and the data were comparable in balance.After treatment,the levels of TCM syn-drome score,LSM,APRI,ALT and GGT in the treatment group were significantly de-creased compared with those before treatment（P<0.05）.Compared with before treatment,the levels of PT,FIB,GGT and IBIL in control group were significantly decreased.After treatment,BUN,HBs Ag and LSM in treatment group were significantly lower than those in control group（P<0.05）.3.（1）Compared with the model group,HXRGD can reduce the degree of inflammation and fibrosis in liver tissue to varying degrees under HE staining.Compared with the mod-el group,Masson staining can reduce the deposition of collagen fiber in liver to the most obvious extent.Compared with the model group,HXRGD could significantly reduce the Ishak score of rats with hepatic fibrosis（P<0.001）.（2）The level of GSH in liver tissue of rats with hepatic fibrosis is significantly decreased,and the expression levels of GSSG,MDA,Fe and Fe2+,and NOX4,NOX4 m RNA and ROS are significantly increased.Com-pared with the model group,HXRGD can significantly increase the level of GSH in liver tissue of rats with liver fibrosis,and decrease the level of GSSG,MDA,Fe,Fe2+,and NOX4,NOX4 m RNA and ROS expression levels（P<0.001）.These results indicated that HXRGD restored the antioxidant capacity of liver to a certain extent,reduced the accumu-lation of lipid peroxides and Fe,and inhibited the NOX4/ROS pathway.4.（1）CCK8 method selected 5%,10%and 20%HXRGD medicated serum as low,medi-um and high dose groups,respectively.（2）In erastin induced ferroptosis activated HSC-T6 cells,Fe,MDA,lipid ROS and GSSG level were all increased,GSH was significantly decreased.ROS,NOX4 and NOX4 m RNA expression levels in activated HSC-T6 cells were significantly increased（P<0.001）.（3）HXRGD high dose medicated serum signifi-cantly reduced GSH in activated HSC-T6,and significantly increased levels of GSSG,li-pid ROS,MDA,Fe,NOX4,NOX4 m RNA and ROS（P<0.001）,which were generally better than Erastin.The results showed that HXRGD induced lipid peroxidation,lipid per-oxide accumulation and Fe deposition in activated HSC-T6 cells.In addition,after the in-tervention of HXRGD medicated serum,the mitochondrial morphology of activated HSC-T6 cells changed,the mitochondria became smaller,the membrane density increased,the mitochondrial ridge was blurred or even disappeared,and the outer membrane of some mitochondria was broken,showing the typical changes of mitochondria when ferroptosis.Conclusions:1.The treatment of hepatic fibrosis of liver-qi and spleen-deficiency type takes"harmony method"as the principle,which not only reflects the treatment principle of soothing the liver and strengthening the spleen,but also highlights the characteristics of harmonizing qi and blood,separating bitter and pungent and simultaneous use of cold and warm.The application of harmony method is flexible and unrestrained,and it is widely used in the treatment of hepatic fibrosis,which is worthy of further discussion.2.HXRGD can effectively reduce hepatic fibrosis,improve symptoms,and reduce ALT level in patients with liver stagnation and spleen deficiency type CHB liver fibrosis,show-ing a good clinical effect.3.HXRGD can reduce the degree of hepatic fibrosis in rat liver tissue and inhibit NOX4/ROS pathway,reduce the consumption of GSH,reduce the accumulation of lipid peroxides,and reduce the accumulation of Fe in the liver,suggesting that HXRGD can down-regulate the NOX4/ROS pathway in the liver of rat hepatic fibrosis and reduce the level of ferroptosis markers.4.HXRGD medicated serum can up-modulate NOX4/ROS pathway,increase the con-sumption of GSH and the accumulation of Fe in activated HSC-T6,increase the accumula-tion of lipid ROS and lipid peroxide,and make mitochondria shrink and show ferroptosis morphological characteristics.These results suggest that HXRGD can promote activated HSC ferroptosis through up-regulation of NOX4/ROS pathway and play an anti-fibrosis role. |
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